Rostro-caudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity

The rostro-caudal extent of odorant receptor expression zones in the rat olfactory epithelium was analysed by means of in situ hybridization. Three broad non-overlapping zones were identified that extended along almost the entire anterior-posterior axis; each zone was composed of several separate bands running anterior to posterior throughout the olfactory epithelium. Superimposed onto these broad zones was the expression area of a particular receptor subtype (OR37); it was restricted to a small region of the epithelial sheet with a high density of reactive neurones in the centre and declining numbers towards the periphery of the region. A quantitative evaluation of the reactive cells revealed that, despite their diferent distribution patterns, all receptor subtypes were expressed in an equal number of neurones.     

Postnatal changes in the ultrastructure of the rat olfactory epithelium: the supranuclear region of the supporting cells

Summary The present electron-microscopical study reports ultrastructural changes occurring in the supranuclear region of the supporting cells of the rat olfactory epithelium during the first 16 days of postnatal life. These changes are concerned with the enclosure of receptor cell dendrites and an increase in the amount of smooth endoplasmic reticulum, which has a specific distribution in the supporting cell. An increase in microvillous projections at the free cell surface is also observed. Moreover, this report demonstrates that the cytoarchitecture of the apical portion of the olfactory epithelium at the 16th day of postnatal life is similar, with respect to the relationship between supporting cells and receptor cell dendrites, to that of adult animals.     

Distribution of histamine in the rat kidney during pregnancy and development

Summary An antiserum against conjugated histamine and two oligonucleotide probes that detect the mRNA encoding L-histidine decarboxylase (HDC) involved in histamine synthesis were used to study the appearance of histamine and its location in the kidneys of fetal, newborn and young postnatal rats and in the kidneys of pregnant rats. On embryonic days 16 and 18 (E16 and E18), some HA-immunoreactive (HA-ir) cells were found within the largest S-shaped bodies. Histamine was found to appear rapidly between the 18th and 20th embryonic days in the convoluted tubules of the kidneys. On postnatal day 0 (P0), the distal convoluted tubules and collecting ducts exhibited bright fluorescence, the intensity of which decreased quickly so that it was faint on day P4 and absent at later stages. In kidneys of pregnant rats HA-ir was found in the epithelium of both the Bowman's capsule, collecting ducts and in a few cells within the tubules. Nonuniform HA-ir was also detected within glomeruli. No evidence for the presence of L-histidine decarboxylase mRNA in kidneys of fetuses or pregnant rats was seen. It is concluded that distinct structures in the developing rat kidney contain histamine during a period around birth from day E20 to day P4. In the pregnant rat, the epithelium that is in direct contact with the urine flow is immunoreactive for histamine from day 16 to 20 of pregnancy. The results suggest that histamine is not synthesized locally in the kidneys but rather originates from other tissues.     

Generation of monoclonal antibodies detecting specific epitopes in olfactory and respiratory epithelia

Summary Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations.     

Immunoreactive galanin-like material in magnocellular hypothalamoneurohypophysial neurones of the rat

Summary Immunoreactive galanin-like material was recently shown to co-exist with vasopressin in parvocellular and magnocellular perikarya of the paraventricular nucleus in the anterior hypothalamus of the rat (Melander et al. 1986). Since this distribution pattern differed from our observation of oxytocin-associated galanin-like immunoreactivity (LI) in the neurohypophysis, we compared in series of 0.5-m thick sections the localisation of galanin-LI with the localisation of oxytocin and vasopressin/dynorphin in the hypothalamus, the median eminence and the neurohypophysis. In the oxytocin system, galanin-LI was intense in oxytocin varicosities of the neurohypophysis. Oxytocin perikarya of the hypothalamic supraoptic and paraventricular nuclei exhibited galanin-LI only after intraventricular injection of colchicine and when sections were treated with trypsin prior to application of the antibody. In the vasopressin/dynorphin system galanin-LI was intense in hypothalamic perikarya after colchicine injection and in neurohypophysial varicosities after treatment of the sections with trypsin. In these neurones, galanin-LI was absent or weak in all elements when treatments with colchicine or trypsin were omitted. Galanin-LI in the neurohypophysis was not co-localised with the numerous fine endings showing GABA-LI. These observations indicate that galanin-like material coexists with vasopressin and oxytocin in the respective magnocellular neurones, although not always in an immunoreactive form.     

Immunohistochemical demonstration of calbindin-containing nerve endings in the rat esophagus

Immunoreactivity for calbindin was found in nerve endings with irregular laminar shapes in the rat esophagus. In the myenteric ganglia, laminar endings of a range of sizes formed a complex network and appeared to lie at the surface of the ganglion. The myenteric ganglia that contained nerve endings were most abundant in the upper portion of the eosphagus, their number decreasing orally to anally. Calbindin-immunoreactive nerve cell bodies were scattered throughout the esophagus. Laminar terminals were found in the connective tissue of the lamina propria immediately beneath the epithelium and in the muscularis mucosae. Occasional nerve branches formed a network of aborizing endings that surrounded part of the submucosal arterioles. Immunoreactive nerve endings in the mucosa and submucosa were present only in the upper part of the cervical esophagus. Unilateral vagotomy caused a remarkable decrease in the number of the myenteric ganglia containing the calbindin-immunoreactive laminar endings after 15 days or survival; in some of ganglia, the laminar structures disappeared and nerve endings showing weak immunoreactivity had an indistinct appearance, so that the outline of the ganglia became obscure. In operated rats at 24 days, the number of innervated ganglia was about half that in normal rats. However, there was no change in the morphology and the occurrence of the immunoreactive laminar structures in the mucosa and submucosa after denervation. The results show that many of the laminar endings that are immunoreactive for calbindin in the myenteric ganglia are derived from the vagus nerve. Thus, the calbindin-immunoreactive nerve endings with laminar expansions that are found in the rat eosphageal wall could be sensory receptors.     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Involution of the rat thymus in experimentally induced hypothyroidism

The thymus, as part of the immune-neuroendocrine axis, is greatly influenced by factors from most endocrine glands, especially the thyroid. Antithyroid drugs (carbimazole and methimazole) were used to induce hypothyroidism in rats. Histological and ultrastructural examination of the thymus showed progressive thymic involution after 4 weeks of drug treatment to the end of observations (7 weeks). The involution was characterised by increased thymocyte apoptosis and thymocyte phagocytosis by macrophages. This resulted in thymocyte depopulation, increases in numbers of interdigitating cells, alterations to mainly subcapsular and medullary epithelial cells, an apparent increase of mast cells and collagen in the capsule and septa, and increased numbers of B cells and plasma cells. Lymphoid cells immuno-reactive with MRC OX12 (which detects B cells) were observed within blood vessel walls, suggesting that they may have been moving in and out of the thymus. The administration of drugs causing hypothyroidism, therefore, also caused marked involution of the thymus.     

Calbindin D-28k-positive neurons in the rat olfactory bulb

Summary We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.     

Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies

Summary The olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3-to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells.     

Pathophysiological role of platelets in acute experimental pancreatitis: influence of endothelin A receptor blockade

The potential pathophysiological role of platelet-endothelium interactions was investigated during ischemia/reperfusion (I/R), and the effect of a selective endothelin(A) receptor antagonist (ET(A)-RA) was evaluated in an acute pancreatitis model. Acute pancreatitis was induced by warm ischemia (60 min) in Wistar rats, and its effects with and without antagonist treatment were investigated. Equivalent sham-operated animals were also studied. Microcirculatory changes were assessed by in vivo microscopy, and serum levels for lipase/amylase and histological specimens were investigated. Capillary constriction to 83.7 +/- 6.7% of sham-operated diameters was observed after 60 min of ischemia. A capillary density of 56.8 +/- 9.3% of the sham-operated group (396.3 +/- 15.8 mm(-1)) was measured after reperfusion. Stagnant leukocytes (329.5 +/- 30.4%) and platelets (337.5 +/- 32.3%) increased in postcapillary venules (P < 0.05). Administration of the ET(A)-RA significantly reduced I/R injury. Capillary diameters were maintained (101.4 +/- 4.5%), and capillary density was improved to 73.3 +/- 7.6% of sham-operated animals (P < 0.05). Stagnant leukocytes (152.3 +/- 10.6%) and platelets (207.1 +/- 19.8%) in sinusoids and postcapillary venules were reduced (P < 0.05). The extent of acute pancreatitis was reduced in the therapy group as indicated by serum lipase/amylase values and histological tissue damage (P < 0.05). Thus, ET(A)-RA therapy was effective in reducing I/R-induced pancreatitis in this experimental model.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunolocalization in lymphoid tissues of the rat

Pituitary adenylate cyclase activating peptide (PACAP) is a novel peptide isolated from the ovine hypothalamus. PACAP exists in 2 molecular forms with 27 (PACAP27) or 38 (PACAP38) amino acid residues. PACAP localization was studied by immunohistochemical methods in central (bone marrow and thymus) and peripheral (spleen, lymph nodes and duodenal mucosa) lymphoid tissues with antisera raised against PACAP27 or PACAP38. PACAP-positive cells were found in all lymphoid tissues examined. These cells were highly positive for PACAP38 but were negative for PACAP27. Morphologically, they were small mononuclear cells with relatively scarce cytoplasm and lymphocyte-like features. PACAP38-positive cells were abundant in peripheral lymphoid tissues (i.e., mesenteric lymph nodes). In the duodenal mucosa, PACAP38-positive cells were located either in the lamina propria or epithelium. These results suggest that PACAP38-positive cells are present within lymphoid tissues and may represent a lymphocyte-like cell subpopulation that has a potential role in cell-to-cell interactions in the immune system and in the integrated communication between neuroendocrine and immune systems.     

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.This work was supported by The Wellcome Trust, UK (CRIG Grant 058122/Z/99/Z/JC/KO), and PEDECIBA, Universidad de la República, Montevideo, Uruguay     

Expression of mRNA of parathyroid hormone-related peptide in fetal bones of the rat

Summary Previous studies have indicated that 19-dayold fetal long bones of the rat contain an adenylyl cyclase-stimulating activity antigenically related to parathyroid hormone-related peptide. To ascertain its origin, Northern blotting and in situ hybridization histochemistry were performed. Results demonstrate that mRNA of parathyroid hormone-related peptide is present in RNA extracted from fetal long bones of the rat and that cells responsible for its production are localized in the periosteum. These cells are not mature osteoblasts because they do not synthesize mRNA of osteocalcin. Thus the present study shows that parathyroid hormone-related peptide could be produced locally, at least in part, in the skeleton of fetal rats.     

Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat

Summary Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.