Showing papers in "Cell and Tissue Research in 1987"

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells.

Abstract: By use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.

Pathway-specific patterns of the co-existence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig.

Abstract: The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin. These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions.

Abstract: N-CAM180, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeletonmembrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM180 by immunoaffinity chromatography and binds specifically to N-CAM180 but not to N-CAM140 or N-CAM120 in a solid-phase binding test. These observations indicate an association of N-CAM180 with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM180 in the surface membrane compared to N-CAM140 (Pollerberg et al. 1986). Together with the fact that N-CAM180 is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM180 in stabilization of cell contacts.

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch.

Abstract: M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.

The ultrastructure of nerve endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands: Identification by an antiserum against a synthetic PDH

Abstract: A high-liter antiserum has been obtained from two rabbits immunized with a glutaraldehyde conjugate of synthetic pigment-dispersing hormone (PDH) from Uca pugilator and bovine thyroglobulin. The antiserum blocked melanophore-dispersing activity of the peptide in vivo. In sinus glands (SG) of Carcinus maenas, Cancer pagurus, Uca pugilator and Orconectes limosus, electron-microscopic immunocytochemistry revealed sparsely distributed axon endings containing a distinct PDH-immunoreactive type of neurosecretory granules (diameter 90–130 nm). Exocytotic figures indicating release of the content of these granules into hemolymph lacunae were occasionally observed. Preservation of fine structure and antigenicity of the PDH granules were markedly dependent on the fixation procedure used. A preliminary experiment with C. maenas showed that preterminal axon dilatations near the basal lamina seemed to accumulate PDH-granules when animals were kept in complete darkness for three days. Immunodot blotting of fractions after high pressure liquid chromatography (HPLC) of extracts from SGs of C. maenas and O. limosus revealed a strongly immunoreactive substance at a retention time very similar to those of synthetic PDHs of Uca pugilator and Pandalus borealis. It is also coincident with a zone of biological activity. Thus, the antigen demonstrated by immunocytochemistry is identical or very similar to one of the known PDHs.

Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice. An autoradiographic study.

Abstract: We test the proposal (McGeachie and Grounds 1985) that myogenesis following severe (crush) injury is prolonged compared with minor (cut) injury. Forty-four mice were injured with a cut and a crush lesion on different legs, and tritiated thymidine was injected at various times after injury (0 to 120 h), samples of regenerated muscle were taken 9d after injury and autoradiography was used to determine the initiation of muscle precursor replication, and duration of proliferation after the two different injuries.

Immunocytochemistry of GABA in the brain and suboesophageal ganglion of Manduca sexta.

Abstract: We have used specific antisera against protein-conjugated gamma-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. About 20,000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-like immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers.

Osteonectin — a differentiation marker of bone cells

Abstract: Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.

Muscle damage and repair in voluntarily running mice: strain and muscle differences.

Abstract: Soleus, extensor digitorum longus and tibialis anterior muscles of mice voluntarily running in wheels for periods of 5 to 120 days were studied in spaced serial and serial cross-sections. Shortly after the onset of running and during the next 2 weeks, degeneration, necrosis, phagocytosis and regeneration of muscle fibers, satellite cell proliferation and cellular infiltration were found in soleus muscles of mice from all strains investigated (CBA/J, NMRI, C57b, NIH, SWS and Balb/c). Tibialis anterior but not extensor digitorum longus muscles were also damaged. Predominantly high-oxidative fibers were affected (both slow-oxidative and fast oxidative glycolytic in soleus, fast-oxidative glycolytic in tibialis anterior). Denervated soleus muscles that had been passively stretched during running were not damaged. Evidence was found that, during the early period of running, split fibers form by myogenesis within (regeneration) or outside (satellite cell proliferation) necrotic muscle fiber segments. Split fibers persisted in solei of long-term (2 to 3 months) exercised CBA/J but not NMRI mice. In 6 out of 20 solei of CBA/J runners exercised for 2 months or longer, fiber-type grouping was observed in the areas where extensive damage usually occurred in the early periods. The results show that different muscles are damaged and repaired to varying degrees and that marked interstrain and inter-individual differences are present. It appears that acute muscle injury occurring upon onset of voluntary running is a usual event in the adaptation of muscles to altered use.

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Abstract: Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.

Regenerating endothelial cells express insulin-like growth factor-I immunoreactivity after arterial injury.

Abstract: In the present study the expression of insulin-like growth factor I (IGF-I; somatomedin C) immunoreactivity was examined in endothelial cells during repair after injury to the intima in the femoral artery of adult rats. Two types of injury were examined: (1) endothelial denudation induced by the use of a catheter, and (2) vessel compression by short-term ligation. In untreated rats, arterial endothelial cells showed no or, only infrequently, low IGF-I immunoreactivity in their cytoplasm. Endothelial cells at the border to the denuded area showed increased IGF-I immunoreactivity one day after injury to the intima of the femoral artery. Thrombocytes and fibrin deposits as well as vital endothelial cells, covered by clots, were immunonegative. The maximal intensity of IGF-I immunoreactivity was reached within 3 days after insult. The IGF-I immunoreactivity in the endothelial cells remained elevated for at least 4 weeks, compared to the controls. Intimai thickenings appeared within a week after injury and many cells in these thickenings showed intense IGF-I immunoreactivity as did the covering endothelial cells. Smooth muscle cells in the media were generally immunonegative during control conditions and after endothelial denudation. Spontaneously hypertensive rats (SHR) showed, similarly to their matched controls (WKY), approximately the same patterns of IGF-I immunoreactivity in their endothelial cells both under normal conditions and after injury. It is concluded that IGF-I is likely to be involved in the repair of the intima in injured arteries.

Sexual dimorphism in neuronal projections from the antennae of silk moths ( Bombyx mori, Antheraea polyphemus ) and the gypsy moth ( Lymantria dispar )

Abstract: The antennal lobe of both sexes of the silk moth Bombyx mori contains 55–60 ventrally located antennal glomeruli; in addition, that of the male contains a dorsal macroglomerular complex (MGC). A group of identifiable glomeruli consisting of two lateral large glomeruli (LLG) and four medial small glomeruli (MSG) is present in both sexes, but the LLG are greatly enlarged in the female. A MGC is also present in the male gypsy moth Lymantria dispar and male giant silk moth Antheraea polyphemus. The MGC in all of these species is organized into 3–4 distinct levels of glomeruli. Antennal sensory fibers were stained by cobalt backfills in B. mori, A. polyphemus, and L. dispar. Most fibers stained from cut long hairs (sensilla trichodea) projected to MGC in males and LLG in both sexes of B. mori. The distribution of fibers in the MGC of B. mori was topographically biased in that a majority of fibers from anterior branches projected medially in MGC while most fibers from posterior branches projected laterally or anteriorly. Terminal arborizations of single fibers were each restricted to a single glomerular level of the MGC. Fibers projecting to the posterior antennal center were frequently stained in cut-hair and control preparations, apparently by uptake of cobalt through intact sensilla on flagellar branches.

Distribution of nerve fibers immunoreactive to neurofilament protein in rat molars and periodontium

Abstract: The distribution of nerve fibers in molars, periodontal ligament and gingiva of the rat shows a complex pattern. Decalcified material including the alveolar bone was sectioned in three different planes and stained by means of immunohistochemistry for detection of the neurofilament protein (NFP); the immunoreactive neural elements were clearly visualized in three-dimensional analyses. NFP-positive nerve fibers formed a subodontoblastic plexus in the roof area of the dental pulp; some of them entered the predentin and dentin directly through the dentinal tubules. This penetration was found mainly in the pulp horn, and was limited to a distance of about 100 μm from the pulpo-dentinal junction. In the periodontal ligament, NFP-positive nerve fibers were found densely distributed in the lower half of the alveolar socket. Two types of nerve terminals were recognized in the periodontal ligament: free nerve endings with tree-like ramifications, and expanded nerve terminals showing button- or glove-like shapes. The former tapered among the periodontal fibers, some even reaching the cementoblastic layer. The latter were located, frequently in groups, within the ligament restricted to the lower third of the alveolar socket. A well-developed plexus of NFP-positive nerves was revealed in the lamina propria of the free gingiva, the innervation being denser toward the epithelium of the gingival crevice. The characteristic distribution of NFP-immunoreactive nerve fibers revealed in this study is discussed in relation to region-specific sensations in the teeth and surrounding tissues.

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Abstract: Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.

Morphology, composition, and function of struts between cardiac myocytes of rat and hamster

Abstract: The morphology, composition, and function of struts that interconnect the lateral surfaces of cardiomyocytes were examined in the hearts of rats and hamsters. Methods included brightfield and fluorescent light microscopy, secondary and backscatter scanning electron microscopy, and transmission electron microscopy in conjunction with silver stain, cationic dye, and antibody to type-I collagen. These studies reveal a twisted, beaded appearance and a complex substructure of collagen fibrils embedded in a ground substance that has a positive reaction with cationic dye. A hierarchy of patterns of branching and attachment was seen among intercellular struts ranging in diameter from 0.1 μm to several urn. The hypothesis that struts tether not only the surfaces but the contractile lattices of laterally adjacent myocytes is supported by the following: (a) the attachments of struts to the collagen weave of the sarcolemma, often lateral to the level of Z bands, (b) the presence of collagen type I in a composite material arrangement, (c) the relative dispositions and configurational changes of struts and myocyte surfaces in various physiological states and induced, non-physiological perturbations of cardiac muscle, (d) the corrugated sarcolemmas with infoldings near Z bands, and (e) the continuity of intracellular filaments from Z bands to the inner aspect of the sarcolemma in relaxed and contracted myocytes. Implications of struts acting as tethers and sites for storage of energy in the motions of myocytes during the cardiac cycle are discussed.

Rapid axoplasmic transport of insulin-like growth factor I in the sciatic nerve of adult rats

Abstract: Somatomedin C (Sm-C; insulin-like growth factor I; IGF-I) is a polypeptide (Mr 7649), often dependent on growth hormone (GH), with trophic effects on several different tissues. Monospecific IGF-I antisera were used to investigate its localization in the sciatic nerve and corresponding nerve cells, as well as its possible axoplasmic transport in the adult rat. IGF-I-like immunoreactivity was demonstrated in anterior horn motor nerve cells in the spinal cord and in spinal- and autonomic ganglion nerve cells. Faint IGF-I immunoreactivity was under normal conditions observed in axons of the sciatic nerve and in the Schwann cells. Using crush technique, accumulation of IGF-I immunoreactivity was seen in dilated axons within 2 h, both proximal and distal to the crush. However, only a small fraction of the anterogradely transported IGF-I immunoreactive material could be demonstrated to be transported in retrograde direction. Colchicine injected proximal to a crush prevented accumulation of IGF-I immunoreactivity proximal to the crush, but not distal to it.

Neuropeptide Y: intrapancreatic neuronal localization and effects on insulin secretion in the mouse.

Abstract: The intrapancreatic localization and the effects on basal and stimulated insulin secretion of neuropeptide Y (NPY) were investigated in the mouse. Immunocyto-chemistry showed NPY to be confined to intrapancreatic nerve fibers mainly associated with blood vessels. Fine varicose NPY fibers were also detected in the exocrine parenchyma and occasionally also within the islets. Double-staining experiments with the use of antisera for both NPY and tyrosine hydroxylase (TH) indicated that most of the NPY fibers were nonadrenergic in nature. Only a population of the NPY fibers occurring around blood vessels showed TH immunoreactivity. Under in vivo conditions, NPY was found to elevate plasma insulin levels slightly when injected intravenously at the high dose level of 8.5 nmol/kg. At lower dose levels, NPY did not affect basal plasma insulin levels, but instead inhibited glucose-induced insulin secretion. Thus, the glucose-induced increment in plasma insulin levels, which was 120±7μU/ml in controls, was reduced to 87 ±5 μU/ml by NPY at 4.25 nmol/kg (p<0.01) and to 98±6μU/ml by NPY at 1.06 nmol/kg (p<0.05). In contrast, the insulin secretory response to the cholinergic agonist carbachol was not affected by NPY. We conclude that NPY nerve fibers occur in the mouse pancreas and that most of these NPY nerve fibers are nonadrenergic. Furthermore, in the mouse, NPY enhances basal plasma insulin levels at high dose levels and inhibits glucose-induced, but not cholinergically induced insulin secretion at lower dose levels under in vivo conditions.

Bone formation in organ cultures of bone marrow.

Abstract: Bone formation in organ cultures of intact marrow fragments from mouse is described. Marrow explants were cultured on the top surface of a millipore filter at a gas-liquid interface. Observations with both light- and electron microscopes demonstrated the formation of a well-organised trabecular matrix lined with osteoblast-like cells. The tissue and cells were positive for alkaline-phosphatase activity. Large amounts of thick, well-banded collagen fibrils and matrix vesicles typical of those found in bone were present. The tissue became mineralised in the presence of 10 mM Na-β-glycerophosphate; in its absence a similar trabecular matrix developed but mineralisation did not take place.

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Abstract: Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.

Effect of retinoic acid on nerve growth factor receptors.

Abstract: Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of 125I-NGF to solubilized receptors. Analysis of 125I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.

The projections of chemically identified nerve fibres in canine ileum.

Abstract: The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.

Trinucleate cells and the ultrastructural localisation of bovine placental lactogen

Abstract: Bovine placental lactogen activity is shown by immunogold electron microscopy to be restricted to the granules and the Golgi body from which they form in the bovine fetal trophectodermal binucleate cell, and granules of similar size and staining reaction in trinucleate "giant" cells found in the maternal uterine epithelium throughout pregnancy. These results support the hypothesis that a fetal binucleate cell forms a maternal giant cell by migration to and fusion with a uterine epithelial cell.

Serotonin-immunoreactive neurons in the antennal lobes and suboesophageal ganglion of the honeybee

Abstract: We have used immunohistochemical methods to investigate the morphology of identified, presumptive serotonergic neurons in the antennal lobes and suboesophageal ganglion of the worker honeybee. A large interneuron (deutocerebral giant, DCG) is described that interconnects the deutocerebral antennal and dorsal lobes with the suboesophageal ganglion and descends into the ventral nerve chord. This neuron is accompanied by a second serotonin-immunoreactive interneuron with projections into the protocerebrum. Two pairs of bilateral immunoreactive serial homologues were identified in each of the three suboesophageal neuromeres and were also found in the thoracic ganglia. With the exception of the frontal commissure, no immunoreactive processes could be found in the peripheral nerves of the brain and the suboesophageal ganglion. The morphological studies on the serial homologues were extended by intracellular injections of Lucifer Yellow combined with immunofluorescence.

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Abstract: A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.

Encapsulation of foreign particles in vitro by separated blood cells from crayfish, Astacus leptodactylus

Abstract: Blood cells from Astacus leptodactylus were separated by density gradient centrifugation and used to study in vitro encapsulation of different objects. Each of the three different populations of haemocytes responded differentially to the foreign particles tested (fungal spores of Aspergillus niger, or ion exchange particles with neutral, positive or negative surface charges). The semigranular cells encapsulated all foreign objects tested, regardless of origin or surface charge, and the capsules developed within 15 h to very dense and tight structures. The granular cells only aggregated around the fungal spores, but no dense capsules were formed. Only a very weak association between granular cells and the beads could be observed. The hyaline cells could not encapsulate any foreign particle, not even if incubated together with granular or semigranular cells. Capsule formation was associated with degranulation of the cells since encapsulation was either inhibited or delayed if the cells were incubated with compounds known to affect degranulation in crayfish granular and semigranular cells, such as A 23187, SITS, EDTA, LPS or laminaran.

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Abstract: The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.

Development of dome epithelium in gut-associated lymphoid tissues: association of IgA with M cells

Abstract: The dome epithelium (DE), which covers gut-associated lymphoid tissues (GALT) and provides both a protective barrier over lymphoid follicles and a route for antigen uptake from the gut, develops in rabbit appendix (caecum) during the first week of neonatal life. To determine if secretory immunoglobulins from maternal milk interact with this developing tissue, their interrelationships in neonatal rabbit appendix were examined by use of immunocytochemical techniques. The glycoprotein, secretory component, was not produced by neonatal rabbits less than 15 days old, since neither the membranous nor the free, secreted forms of maternal secretory component were associated with villi or DE of neonates. Immunoglobulin A (IgA), but neither IgG nor IgM, were noted on DE by light microscopy, even though IgG was abundant in the villus lamina propria and vascular spaces. The epithelial IgA was distributed, in a patchy pattern, across the upper dome surface of some two-day-old, and all five-and ten-day old nursing animals, but IgA was not on DE of rabbits prevented from nursing. Immuno-electron microscopy of appendix from nursed rabbits revealed IgA directly over the apical surface of M cells, where it formed a continuous, thick coating without binding to adjacent immature absorptive cells; it was also within apical vacuoles of M cell cytoplasm. The distribution of IgA on the DE of rabbit appendices indicated that in differentiating GALT, maternal IgA reacted preferentially with M cells or pre-M cells, leading to speculation concerning a role for IgA in the development of GALT and in establishment of mucosal immune responses in neonates.

A model of myogenesis in vivo, derived from detailed autoradiographic studies of regenerating skeletal muscle, challenges the concept of quantal mitosis.

Abstract: We have recently shown that myogenesis following severe injury is prolonged compared with minor injury (McGeachie and Grounds 1987). In this previous autoradiographic study 44 mice were injected with tritiated thymidine at various times after muscle injury (0 to 120 h), and samples were taken 9d after injury to determine the percentage of labelled myotube nuclei. In the present study the same experimental data are analysed in detail to reveal how many times labelled muscle precursors divided before fusing to form myotubes.

Origin of regenerating Leydig cells in the testis of the adult rat. An ultrastructural, morphometric and hormonal assay study.

Abstract: Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (approximately 1-2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (approximately 15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni.

Abstract: The peroxidase-antiperoxidase immunocytochemical procedure was used to study the distribution of ovine corticotropin-releasing factor (CRF) and arginine vasotocin (AVT) immunoreactivities sequentially in the same sections or in adjacent sections of the brain and pituitary of Catostomus commersoni. It was found that all CRF-immunoreactive (IR) neurons in the nucleus preopticus (NPO) also contained AVT immunoreactivity. Co-localization of both immunoreactivities was also observed in fibres forming the preoptic-pituitary tract and in the neurohypophyseal digitations, the IR-CRF and IR-AVT fibres projecting mainly to the neurointermediate lobe (NIL) of the pituitary. An additional population of exclusively IR-AVT neurons and fibres in the NPO, preoptic-pituitary tract and NIL was also observed. Exclusive CRF-immunostaining was found in neurons of the nucleus lateralis tuberis (NLT), in fibres distributed in some diencephalic nuclei and in the neurohypophyseal digitations in the region of the rostral pars distalis (RPD). These results suggest (i) that CRF- and AVT-like substances, present in NIL fibres (probably originating in the NPO), may have an integrated role in the release of the cell products from the pars intermedia, and (ii) that the control of corticotrops in the rostral pars distalis, innervated exclusively by IR-CRF fibres (probably originating in the NLT), does not require a simultaneous presence of CRF- and AVT-like substances.