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The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4-10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvements in the differentiation of heart, skeletal and neuronal structures.

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Development of trophic interactions in the vertebrate peripheral nervous system.

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of the trk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.

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Occurrence of myelin-associated glycoprotein (MAG)-like immunoreactivity in some nervous, endocrine, and immune-related cells of the rat. An immunohistochemical study.

The occurrence and distribution of myelin-associated glycoprotein (MAG)-like immunoreactivity was investigated in the rat using a polyclonal antibody to MAG purified from rat brain. In the nervous system, MAG immunoreactivity was found in the periaxonal portion of the myelinated fibers and in a small number of oligodendroglia in the cortex, hippocampus, and the spinal cord. The sheath of Schwann cells in unmyelinated fibers and satellite cells in the spinal ganglia were also immunoreactive for MAG. In the endocrine system, the noradrenaline-containing cells in the adrenal medulla and some endocrine cells in the duodenum showed MAG immunoreactivity. In the immune system, numerous reticular cells with slender cytoplasmic processes, which formed a dense network, were immunopositive for MAG within the germinal center in the lymph nodes and spleen. In the thymus, a number of epithelial reticular cells within the medulla showed variation in staining intensity. These findings provide new information on the wide distribution of MAG immunoreactivity in the nervous, endocrine, and immune systems, and may contribute to the further understanding of the biological roles of this protein.

Adrenal Glands↗

Skin-visceral divergent projection of cholecystokinin-containing dorsal root ganglion neurons: a tri-labelling study with fluorescent tracers and immunohistochemistry.

Skin-visceral divergent projections of cholecystokinin (CCK)-containing dorsal root ganglion neurons were studied by combined technique of fluorescent double-labelling and immunohistochemistry. Fast blue (FB) and nuclear yellow (NY) were injected into the coeliac ganglion and the cutaneous branches of left 9th-11th intercostal nerves, respectively. Three kinds of neurons labelled with fluorescein were observed in T9-11 dorsal root ganglia: FB-labelled neurons with blue-fluorescent cytoplasm; NY-labelled neurons with yellow-fluorescent nucleus and double-labelled neurons with blue cytoplasm and yellow nucleus. The double-labelled neurons were found to account for 2.8% of total labelled neurons. The sections containing neurons labelled with fluorescein were stained by CCK-immunohistochemical procedure. Four kinds of neurons could be identified: NY-neurons with CCK-immunoreactivity (NY+CCK); FB-neurons with CCK-immunoreactivity (FB+CCK); NY+FB neurons with CCK-immunoreactivity (NY+FB+CCK); and neurons only CCK-positive. NY+FB+CCK tri-labelled neurons accounted for approximately 11.5% of NY+FB double-labelled neurons, and for 0.4% of all CCK-positive neurons. The findings clearly indicated that the peripheral processes of some sensory dorsal root ganglion neurons divergently project to both skin and visceral structure and contain CCK.

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The expression of Quox 1, a homeodomain-containing protein, in sympathetic ganglion cells is regulated in vitro by growth factors.

Quox 1, a quail homeobox gene, is the first vertebrate Antp-type homeobox gene to be described that is expressed in the forebrain. We have already shown that the Quox 1 protein is specifically expressed in post-mitotic sensory neurons. A subpopulation of sympathetic ganglion cells was also found to be labelled by anti-Quox 1 in vitro, but it is not clear whether this protein is expressed in sympathetic ganglion cells in vivo and, if so, the conditions which regulate its expression in vitro. In the present study, we used immunocytochemistry to find out whether Quox 1 expression in sympathetic ganglion cells in vitro is regulated by environmental signals. We found that several peptide growth factors can regulate Quox 1 expression in cultured sympathetic ganglion cells, and that they do so at physiological concentration and in a variety of ways. Basic fibroblast growth factor (FGF-2) induces Quox 1 protein expression, whereas insulin and human insulin-like growth factor-I (IGF-I) down-regulate Quox 1 expression.

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Cytological changes during bioluminescence production in dissociated photocytes from the ophiuroid Amphipholis squamata (Echinodermata).

Bioluminescence in the ophiuroid Amphipholis squamata is produced by photocytes located within the spinal ganglia of arm spines. Ganglionic cells were dissociated (pronase digestion) and photocytes separated from other cell types by using a continuous density Percoll gradient. Aliquots from a stock suspension of photocytes in artificial sea water were stimulated to produce light by using KCl or acetylcholine and fixed for ultrastructural observation at different times of the luminous process. Preluminescent, luminescent, and postluminescent photocytes contained various intracytoplasmic structures, such as Golgi, flat and distended rough endoplasmic reticulum cisternae, bundles of fibrils, and up to six types of membrane-bounded vesicles. These structures either co-occurred or succeeded one another during the process of light production, indicating that they were most probably participating in the luminescence reaction. Two types of vesicles, sharing some ultrastructural features, probably represented the microsources of the photocytes. One type occurred almost exclusively in luminescent photocytes, and the other almost exclusively in postluminescent photocytes, suggesting that one may be transformed into the other. The latter type of vesicle contained densely packed fibro-tubular units, giving a characteristic paracrystalline appearance to postluminescent photocytes.

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The retrograde transport of horseradish peroxidase from the developing limb of the chick embryo.

Chick embryos ranging in age from 4.0 to 18 days of incubation and 1-2-day-old hatchlings received injections of HRP solutions directly into the leg musculature. After survival periods of from 5 to 25 h motoneurons and cells in the spinal sensory ganglia were found to be stained with the HRP reaction product. It was found that the first appearance of a positive HRP reaction coincided with the time when nerve processes are first detected in the limb-bud by silver techniques (i.e. at 4.5 days of incubation). Only neurons with processes in the region of injection showed a positive reaction. The demonstration of a retrograde transport mechanism in neurons and axons which are still undergoing growth and differentiation provides a possible mechanism for the peripheral regulation of certain features of CNS neurogenesis. The application of this technique during development may also allow one to map neuroanatomical pathways during their formation.

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Reduction by reserpine of the accumulation of retrogradely transported [125I]nerve growth factor in sympathetic neurons.

Experiments were carried out to determine if stimuli which augment preganglionic nerve activity to sympathetic neurons, and thereby cause trans-synaptic induction, increase the retrograde transport of nerve growth factor (NGF). It was found that nerve activity had no effect on retrograde transport of [125I]NGF. It was found, however, that reserpine decreased retrograde transport of [125I]NGF and this inhibition was characterized. Reserpine decreased the maximal accumulation of intravenously administered [125I]NGF in superior cervical ganglia (SCG) by about 60%. It also caused a distinct shift in the time course of accumulation so that maximal accumulation was seen 12 h after [125I]NGF injection rather than at 9 h as in control animals. Reserpine had no effect on retrograde transport in sensory neurons. Dose--response curves showed that maximal inhibition occurred with doses of reserpine of 2.5 mg/kg i.p. and that reserpine was not able to completely block transport at any dose. The maximal inhibition of retrograde transport was achieved within 30 min of reserpine administration and inhibitory activity was unchanged for 36 h. The ability of sympathetic neurons to transport [125I]NGF subsequently recovered and was normal 96 h after reserpine administration. The inhibitory effect of reserpine appears to be due to an action at or very near to the nerve terminal since it was effective at reducing NGF transport at very low doses (0.33 microgram) when co-administered directly into the eye with [125I]NGF. An action of reserpine at the nerve terminal was further suggested by the inability of reserpine to affect transport if the drug was given 4 h after [125I]NGF administration. Based upon these data, it is suggested that there may be two pools of retrogradely transported NGF and that only more rapidly turning over pool is reserpine-sensitive. This pool may represent the retrogradely moving synaptic vesicles or some derivative of the vesicles.

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Correlation with superior cervical sympathetic ganglion and sympathetic nerve innervation of intracranial artery-electron microscopical studies.

When the superior cervical ganglion was resected in dogs, nerve degeneration in arterial walls began after about 28 h and marked degenerative substance was shown after 40-48 h; after 4 days the small cored vesicles of adrenergic axons disappeared. The same condition was seen after 3 months, but after 6 months the small cored vesicles were again visible. When the middle cerebral artery was examined by separating it into the perforating artery near to the internal carotid artery and the peripheral portion of the middle cerebral artery, degeneration of the nerve fibers of the arterial walls occurred earlier in the more proximal portion. The distribution of adrenergic nerve fibers from the superior cervical ganglion is bilateral in the anterior cerebral artery from the anterior communicating artery to the peripheral region, basilar artery, and vertebral artery, but ipsilateral only in the anterior cerebral artery as far as the anterior communicating artery, middle cerebral artery, posterior communicating artery, posterior cerebral artery and superior cerebellar artery. Degeneration of nerve fibers of the walls of these cerebral arteries was not seen ever after stellate ganglionectomy in both sides.

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Time and dose dependencies of effects of nerve growth factor on sympathetic and sensory neurons in neonatal rats.

Nerve growth factor (NGF) plays a role in the development of several components of the sympathetic and sensory nervous systems. The objectives of this study were to examine the time and dose dependencies of some of the well known effects of NGF on sympathetic ganglia and to examine qualitatively and quantitatively the recently described effects on sensory ganglia of neonatal rats. Single doses of NGF as low as 0.1 mg/kg produce increases in tyrosine hydroxylase (TOH) activity in superior cervical ganglia (SCG), and doses of 3 mg/kg produce maximal effects. Larger doses and longer treatments are required to see increases in protein content of the SCG. Larger doses are also required to affect TOH activity in the adrenal gland. Increases in TOH activity in SCG can be observed within 18 h of injection. Chronic NGF treatment for three weeks produces no change in blood pressure or heart rate in neonatal rats. chronic administration of NGF (1 or 3 mg/kg/day) results in dose-related increases in the protein content of dorsal root ganglia (DRG). The increase in protein content of the DRG was associated with an increase in the diameter of smaller neurons (those less than 30 micron in diameter), but NGF caused no change in the number of neurons.

Adrenal Glands↗

A slow EPSP in mammalian inferior mesenteric ganglion persists after in vivo capsaicin.

The non-cholinergic slow excitatory postsynaptic potential (EPSP) in the guinea pig inferior mesenteric ganglion (IMG) is believed to be mediated by substance P (SP). Yet, in ganglia of guinea pigs treated with capsaicin (50, 100, or 350 mg/kg, s.c.), a compound that depletes SP from sensory neurons, slow EPSPs were only 50% smaller in amplitude than those in vehicle-treated controls; this was true at all dosages. When ganglia from capsaicin-treated animals were desensitized to SP, slow EPSP amplitude was decreased by 50% from predesensitization values. These data indicate that capsaicin does not eliminate the slow EPSP in the IMG and suggest that both SP and another transmitter are involved in co-mediating this response.

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Astrocyte growth enhanced by culture supernatant of a cloned rat thymic myoid cell.

A conditioned medium (CM) from a cloned rat thymic myoid cell (IT-45R92) was used to investigate a possible relationship between the thymus and brain. CM stimulated the DNA synthesis of the cultured perinatal rat brain cells and spleen myelo-lymphoid cells but not that of the other types of cells including dorsal root ganglia, spinal cord, heart muscle, skeletal muscle, kidney, liver, skin, and thymic fibroblasts. CM appeared to stimulate glioblasts to differentiate into mature astrocytes that have a characteristic intracellular fibrous structure and a glial fibrillary acidic protein. These results indicated that thymic myoid cells release (a) 'cytokine(s)' for the astrocytes and myelo-lymphoid cells, similar to a 'lymphokine' (glia cell stimulating factor, GSF) produced by murine and human lymphocytes stimulated with mitogens.

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The use of the regenerating frog sciatic nerve for pharmacological studies of orthograde and retrograde axonal transport.

The outgrowth region of the regenerating frog sciatic nerve shows an increased permeability for various drugs, which has been utilized for pharmacological studies of axonal transport. Six days after a bilateral crush lesion, the nerves, including the spinal ganglia, were incubated in a compartmented chamber. Orthograde transport was assessed from the proximodistal distribution and the accumulation of labelled proteins in the nerve growth region. Retrograde transport was examined by allowing orthogradely transported materials to reverse at the regenerating region and then to accumulate at a ligature during a second incubation period. The distribution of radioactivity along the nerve was assayed by fluorography of whole-mount nerve preparations or by scintillation counting. Fluorography made it possible to increase the spatial resolution and to demonstrate effects in the elongating part of the regenerating nerve. Colchicine at low concentrations (10-100 microM) only inhibited axonal transport in the outgrowth region (6 mm long at 6 days after crush) and along some mm of the nerve proximal to the crush. Compound 48/80 (50 micrograms/ml), the most specific calmodulin inhibitor so far described, inhibited the transport along the same part of the nerve. Cytochalasin B (10 micrograms/ml) inhibited transport by effects limited to the outgrowth region. Both orthograde and retrograde transport showed sensitivity to these and some other drugs. The regenerating frog sciatic nerve seems to have significant advantages for pharmacological studies of axonal transport.

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Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion.

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.

Animals↗

Selective distribution of the 66-kDa neuronal intermediate filament protein in the sensory and autonomic nervous system of the guinea-pig.

The immunohistochemical distribution of a recently identified 66-kDa neurofilament protein (NF-66) was investigated in peripheral and autonomic ganglia of the guinea-pig where it has been previously established that other neuronal intermediate filament proteins have a selective distribution. NF-66 immunoreactivity was observed in distinct subpopulations of neurons and did not coexist completely with either the neurofilament triplet or a 57-kDa intermediate filament protein (peripherin). NF-66 labelling was identical to that observed with an antibody to a 150-kDa intermediate filament or associated protein (CH1). These results further demonstrate that different neuronal intermediate filament proteins are present in selective subpopulations of neurons and that these proteins are, therefore, likely to have cell type-specific roles.

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Distribution of calretinin mRNA in rat spinal cord and dorsal root ganglia: a study using non-radioactive in situ hybridization histochemistry.

Using non-radioactive in situ hybridization calretinin mRNA was detected in numerous small neurons within lamina II and IV of the dorsal horn. Many labelled cells are distributed over the whole ventral horn; however, no motoneurons contained the mRNA. In dorsal root ganglia 4.9 +/- 1.7% (mean +/- S.D., n = 5 animals) of the primary afferent neurons contained calretinin mRNA. Labelled cells were of intermediate and large size with diameters ranging from 36 to 68 microns indicating that calretinin is synthesized in neurons with myelinated afferent fibers and presumably a corpuscular ending.

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