Nerve growth factor receptor (NGFR) immunoreactivity in skeletal muscles of the rat.

The peroxidase-antiperoxidase (PAP) method, and a specific monoclonal antibody (192-IgG) were used to determine the localization of nerve growth factor receptor (NGFr) in the skeletal muscles of the adult rats. The rectus femoris and the gastrocnemius (medialis and lateralis) muscles were analyzed. Occurrence of NGFr immunoreactivity was observed in: 1) a subpopulation of myelinated nerve fibers within muscle nerve trunks; 2) the vascular adventitia and nerve-like profiles around the blood vessels; 3) the outer capsule and the surface of the intrafusal muscle fibers of muscle spindles. Conversely, images, suggesting the presence of NGFr on muscle fibers or in motor end-plates, were not found. Our results suggest the presence of NGF-binding sites in sensory and sympathetic nerve fibers, and/or their target tissues localized on the skeletal muscles of the rat, whereas the motor nerve fibers lack of NGFr. The dependence of sympathetic neurons, proprioceptive primary sensory neurons, and motoneurons innervating the mammalian muscles upon NGF or other neurotrophic factors is discussed.     

Expression of nerve growth factor receptor immunoreactivity in the rat choroid plexus.

The presence and localization of nerve growth factor receptors (NGFr) in the choroid plexus of the adult rat has been investigated immunohistochemically using an anti-rat NGFr monoclonal antibody (192-IgG). A moderate to strong immunoreaction was observed in the epithelial cells of the choroid plexus, whereas the choroidal blood vessels and connective tissue remained unlabelled. Moreover, no sex-differences were encountered in the NGFr immunoreaction intensity and Bouin fixative was more effective than 10% formaldehyde evidenciating the NGFr immunostain. Occasionally, ependymal cells displaying NGFr immunoreactivity were observed. Present data demonstrate that the choroid plexus of the rat contain NGFr, probably low-affinity NGFr, and suggest an involvement of NGF in the regulation of cerebrospinal fluid secretion, but the importance of these findings, if any, must be investigated in future studies.     

Nerve growth factor receptors in the peripubertal rat ovary.

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain.

We have localized acidic fibroblast growth factor (aFGF) mRNA in the developing and adult rat brain using in situ hybridization histochemistry. Prenatally, hybridization to aFGF mRNA was observed throughout the brain, with the strongest signal associated with cells of the developing cortical plate. Postnatally, labeling was localized to specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer and dentate granule cells was observed and became progressively more intense with maturation. Labeling was also observed in both the external and internal granule cell layers of the developing cerebellum. Pyramidal cells of the neocortex as well as neurons of the substantia nigra and locus ceruleus also express aFGF. This pattern persists into adulthood, although the intensity of the labeling is significantly reduced in the adult brain. These patterns of hybridization correlate with specific developmental events and suggest that aFGF plays a significant role in both central nervous system development and neuronal viability in the adult brain.     

Differences in postmortem stability of sex steroid receptor immunoreactivity in rat brain.

Difficulties in demonstrating sex steroid receptors in the human brain by immunohistochemistry (IHC) may depend on postmortem delay and a long fixation time. The effect of different postmortem times was therefore studied in rat brain kept in the skull at room temperature for 0, 6, or 24 hr after death. After a long fixation for 20 days, hypothalami were embedded in paraffin and sections were immunohistochemically stained for androgen receptor (AR), estrogen receptor-alpha (ER), or progesterone receptor (PR). Retrieving the antigenic sites by microwave pretreatment was essential to obtain successful IHC in all groups studied. In general, immunoreactivity was restricted to the cell nuclei. However, the intensity of the staining appeared to be strongly dependent on the different receptor antigens and postmortem time. Both AR and ER but not PR immunoreactivity were decreased after immersion-fixation compared to the perfused sections at time point zero. In brains fixed by immersion, all three receptors decreased gradually with increasing postmortem time, and ER became hardly detectable after 24 hr postmortem. The results of these experiments show that, with the protocol used, postmortem variables and lengthy fixation do not, in principle, prevent sex steroid receptor IHC in human material. The outcome of the immunostaining, however, might be strongly dependent on the epitopes and/or antibody used.     

Nerve growth factor receptor-like immunoreactivity in primary and permanent canine tooth pulps of the cat

Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.     

Estrogen receptor immunoreactivity in Schwann-like brain macroglia.

Olfactory ensheathing cells, tanycytes, pituicytes, pineal glia, retinal Müller cells, and Bergmann glia of normal male rats express concomitantly estrogen receptor, low-affinity neurotrophin receptor, antigen O4, and GFAP, markers characteristic of nonmyelinating Schwann cells. These cells were able to survive and proliferate when cultured from adult tissue, promoted neurite outgrowth, and could guide and ensheath growing neurites. We called this distinct group of growth-promoting central nervous system (CNS) macroglia aldynoglia (Greek: to make grow). Its proliferative and growth-promoting properties seem to be retained during the whole lifetime of the organism in those CNS loci where normal function depends on continuous axon renewal. Aldynoglia plasticity seems totally or partially lost with age where and when it is no longer critical, as in the case of adult cortical and spinal cord radial glia. The concomitant expression of estrogen receptor and low-affinity neurotrophin receptor may promote Schwann-like plasticity of glial cells.     

Gut-type glucagon immunoreactivity in nerves of the rat brain.

Nerve fibers reacting with antisera demonstrating gut-type glucagon were numerous in certain areas of hypothalamus and thalamus but absent from neocortex and hippocampus. They did not react with glucagon antisera specific for pancreatic type glucagon. Immunoreactive cell bodies were not observed.     

Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis

Introduction  

We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment. Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF).     

Epidermal growth factor receptor internalization and biosynthesis in the diabetic rat.

The number of surface EGF receptors as well as their internalization rate and biosynthesis were analyzed in hepatocytes freshly isolated from control, streptozotocin-diabetic, and insulin-treated diabetic rats. All three parameters were decreased in diabetic animals and values were corrected by insulin treatment. Moreover, the inhibition of synthesis was specific for the EGF receptor since the other biosynthetically labeled proteins were not affected. These data demonstrate that the reduced number of hepatocyte surface EGF receptors results from an inhibition of EGF-receptor synthesis which is not compensated by a reduced internalization rate.     

Distribution of beacon immunoreactivity in the rat brain

Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.     

Nerve growth factor chromatin receptor and cell surface receptor-regulated growth of melanocytes and nevus cells.

RNA synthesis in melanocytes and nevus cells, and the proliferation of those cells in the presence of nerve growth factor (NGF) and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), were found to correlate with the amount of NGF bound to the chromatin versus the total internalized NGF (n/c NGF = nuclear/cellular ration). In nevus cells and in melanocytes of the early passages (n/c NGF = 0.16-0.18), NGF slightly activated RNA synthesis but was without any effect on cell growth. At passage 5-6 of melanocytes (n/c NGF = 0.88), NGF inhibited RNA synthesis, which led to inhibition of cell growth. Removal of TPA from the culture of nevus cells resulted in increased n/c NGF ratio and in a switch from activatory to inhibitory action of NGF. The possibility that the cell surface receptor mediated the stimulatory effect of NGF and may antagonize the chromatin receptor-mediated inhibitory effect of NGF of melanocyte and nevus cell growth is discussed.     

Nerve growth factor receptor from rabbit sympathetic ganglia membranes. Relationship between subforms

The receptor for nerve growth factor (NGF) was purified from Triton X-100 extracts of sympathetic ganglia membranes by affinity chromatography on NGF-Sepharose. Elution of purified receptor was accomplished at pH 5 in the presence of 1 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the purified iodinated receptor showed three major bands at Mr = 126,000, Mr = 105,000, and Mr = 81,000. Affinity labeling of the purified receptor using 125I-NGF and the photoreactive agent N-hydroxysuccinimidyl-p-azidobenzoate resulted in two major cross-linked complexes corresponding to Mr = 135,000 and Mr = 110,000. This labeling pattern is similar to that observed with sympathetic ganglia membranes (Massague, J., Guillette, B. J., Czech, M. P., Morgan, C. J., and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9419-9424) and indicates that these two forms do not arise from the cross-linking procedure. Reaction of the photoaffinity labeled NGF receptors with increasing amounts of trypsin resulted in a progressive decrease in the high molecular weight complex with a concomitant increase in the low molecular weight form. When the larger complex was isolated by electroelution from a sodium dodecyl sulfate gel and treated with trypsin, a species corresponding to Mr = 100,000 was generated. These observations are best explained by a precursor-product relationship for the two NGF receptor species of sympathetic neurons.     

EphB3 receptor and ligand expression in the adult rat brain

Summary Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.     

Immunohistochemical localization of insulin-like growth factor I in the adult rat

Summary Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradeca peptide, representing the carboxyterminal amino acids 57–70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I.High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoetic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoeitic, trombocytopoeitic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine.The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation. Certain cells involved in secretory processes also displayed high IGF-I immunoreactivity. The wide distribution of IGF-I indicates that the circulating pool of IGF-I has multiple origins.     

Nerve growth factor preserves a critical motor period in rat striatum

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.