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BACKGROUND: Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. RESULTS: Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. CONCLUSIONS: Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

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The development of the adrenal gland in the lizard Calotes versicolor was studied histologically and histochemically from the day of oviposition (stage 27) to 60 days after hatching. At stage 27, the adrenocortical cells are found in association with the genital ridge (primordial gonad). The separation of adrenocortical cells from the gonad takes place at stage 31. Organization of adrenocortical cells into cords takes place at stage 34. The catecholamine-secreting chromaffin cells can be seen distinctly on the dorsal region of the adrenal at stage 36, indicating the presence of biologically active catecholamines; the noradrenaline-secreting chromaffin cells appear first at stage 36 and the adrenaline-secreting cells appear later at stage 41. The cortico-medullary ratio of 6:1 during early embryonic development decreases with the increase in age and is 3:1 in posthatching lizards. The histochemical localization of Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase in the adrenocortical cells as early as at stage 27 (prior to the gonadal differentiation) indicates the capability of these cells to synthesize steroids. The intensity of the enzyme activity is maximum on the day of hatching and remains more or less the same in the posthatching lizards. The localization of 17beta-HSD enzyme activity observed in the adrenocortical cells at stage 34 is suggestive of their ability to synthesize sex steroids during embryonic life. The intense 3beta-HSD activity on the day of hatching in C. versicolor suggests high production of steroids which may be corticoids. The results of the present work also suggest that the onset of steroid secretion occurs prior to catecholamine secretion during embryogenesis of the adrenal gland in C. versicolor. In addition, there is a significant relationship between ontogenic steroidogenesis of the adrenal gland and sexual differentiation of the gonad.

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We have studied the contribution of proliferation and hypertrophy of glandular cells to the ontogenetic growth of adrenal chromaffin tissue using several methods (organometry, cytometry, cytophotometric quantitation of DNA in the nuclei, radioautographic analysis of 3H-thymidine incorporation, calculation of the mitotic index and proportion of binuclear cells, as well as stereological analysis). Mitotic division of diploid glandular cells is the main cellular mechanism of postnatal growth of chromaffinocytes. It is most prominent during the first 2 weeks of life and is maintained at a rather high level throughout the life of animals (the daily proliferative pool in 6-month-old and 30-month-old rats equals 0.3%). Development of cellular hypertrophy has been noted during the first 6 months after birth. The population of chromaffinocytes throughout life is practically diploid; the proportion of tetraploid (binuclear) cells does not exceed 1-1.5%. The growth of adrenal chromaffin tissue during the first month of life is generally supported by hyperplasia and hypertrophy of norepinephrocytes and later of epinephrocytes. The contribution of cell proliferation and hypertrophy to postnatal growth of each subpopulation appears to be equal.

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The aim of these examinations was to determine the influence of dexamethasone (Dx)-treatment of gravid females, on day 16 of gestation on the development of medullary chromaffin tissue of their fetuses and neonatal offspring. In conducting these investigations we used stereological as well as spectrofluorimetric measurements, in 20-day-old fetuses and 1-, 3-, 5-, 7-, 9-, 11-, 13- and 14-day-old neonatal rats. Single Dx-treatment (1.5 mg/kg bw) of the dams led to a significant decrease in body and adrenal weight of their fetuses and neonatal offspring, and also reduction of the medullary volume and the number of chromaffin cells during the entire period examined as a result of decreased cell proliferation in the fetal and early neonatal period (till the 5th day of age). The proliferative activity of the chromaffin cells was evaluated through the mitotic index after applying the cytostatic vincristine-sulphate. During the second neonatal week the mitotic index showed significantly higher values in comparison with the corresponding controls, which indicates that there is regeneration and recovery of the adrenal gland medulla. Adrenaline content in the adrenal gland tissue of offspring of Dx-treated dams was significantly reduced only on the 1st neonatal day. Thus, the change in blood glucocorticoid level of pregnant females after a single Dx injection during the period critical for development of the hypothalamo-pituitary-adrenal system in fetuses affects the development and kinetics of medullar chromaffin cell division.

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We have investigated the regulation of the morphological phenotype of chromaffin cells cultured from 6-day-old rat adrenal glands. We show that pituitary adenylate cyclase activating polypeptide (PACAP), which is present in and released from nerves innervating chromaffin cells, rapidly induces neuritic growth, affecting 25% of tyrosine hydroxylase-positive chromaffin cells after 3 days at an optimal concentration of about 20 nM. PACAP does not synergistically act with other factors known to promote neurite growth, including nerve growth factor (NGF), basic fibroblast growth factor (bFGF, FGF-2), and ciliary neurotrophic factor (CNTF). The neurite promoting effect of PACAP and FGF-2 is entirely overridden by dexamethasone (2 x 10(-8) M) suggesting that, despite the presence of these promoting factors in the adrenal medulla, glucocorticoids from the adrenal cortex are probably sufficient to prevent the development of neuronal traits in adrenal chromaffin cells.

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The chromaffin cells of the adult rat adrenal medulla are essentially growth arrested in situ, but can proliferate in vitro, suggesting the existence of growth inhibitory factors in the adrenal gland. We have investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) could be involved in the growth arrest of adrenal chromaffin cells. In adult rat adrenal gland, PACAP38 was detected by radioimmunoassay and high-performance liquid chromatography and its concentration in the medulla was estimated as 24 nmol/kg wet tissue. Immunohistochemistry of the neonatal and adult rat adrenal medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0.5 nM, respectively, as demonstrated by bromodeoxyuridine pulse-labeling and immunocytochemical staining of cell nuclei. In comparison, corticosterone inhibited neonatal and adult chromaffin cell proliferation by 70% and 95%, respectively, with half-maximal effect at 100 nM. In neonatal chromaffin cells, 100 nM PACAP38 and 1 microM corticosterone added together abolished proliferation completely (99.8% inhibition). Finally, PACAP38 increased cell survival but showed little neurite-promoting activity in the chromaffin cells. Our data suggest that neurally derived PACAP38, in conjunction with glucocorticoids, may override growth factor mitogenic signals, leading to the postmitotic state of chromaffin cells in the adult adrenal medulla.

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We investigated the effects of nerve growth factor (NGF) and glucocorticoid on the differentiation of rat chromaffin cells during development (postnatal day 0, 5, 10, 15, 25) in vitro, by evaluating four parameters of cytological alterations; the percentage of process-bearing cells, the number of primary processes, the number of branches per cell and the total length of processes. Cultures of dissociated chromaffin cells from each developmental stage were maintained in medium containing a combination of NGF and/or the synthetic glucocorticoid, dexamethasone (DEX). All parameters for neuronal differentiation were highest in chromaffin cells from P0 animals when incubated in NGF(+)/DEX(-), but lowest in those from animals at P25 incubated in NGF(-)/DEX(+). In addition, chromaffin cells became progressively insensitive to the effects of NGF on cytological differentiation from P0 to P25. These results suggest that as development proceeds locally-derived factors become less potent on chromaffin cell differentiation.

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The developmental expression of neuropeptide Y (NPY) and leucine-enkephalin (L-Enk) was examined in embryonic, early postnatal, and adult chromaffin cells with double- and triple-label immunocytochemical techniques and compared to the expression of immunoreactivity for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). In addition, the establishment of preganglionic innervation was assessed by labeling for choline acetyltransferase (ChAT) and L-Enk. NPY-IR was detectable on embryonic (E) day 15 in a clustered subpopulation of TH-IR cells. L-Enk and PNMT-IR cells were initially present on E16 in a separate nonclustered population of TH-IR cells. By late embryonic development, twice as many TH-IR cells expressed NPY and 4 times as many expressed L-Enk as in the adult. In contrast to early embryonic development, NPY-IR was evident in both the clustered and nonclustered subpopulation of TH-IR cells at this time. The proportion of NPY-IR chromaffin cells decreased to adult values during the first postnatal week at the time when obviously clustered TH-IR cells were no longer observed. The embryonic rise in the proportion of L-Enk-IR cells correlates with the developmental increase in glucocorticoid production, while the postnatal decrease corresponds to the appearance of ChAT-IR in the preganglionic innervation of the adrenal medulla. These results indicate that NPY and L-Enk are expressed at different times and in different subpopulations of cells in the embryonic adrenal. Further, the observation that peptide expression by chromaffin cells undergoes marked changes during development raises the possibility that a number of factors including developmental history, environmental signals and impulse activity play a role in the regulation of neuropeptide expression in sympathoadrenal derivatives of the neural crest.

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The authors previously evaluated the expression of a panel of chromaffin-related genes during histogenesis of the human adrenal medulla. In these studies, chromaffin and nonchromaffin adrenal neuroblasts were identified. To better characterize these nonchromaffin neuroblasts, the authors evaluated two additional markers: HNK-1, an antibody recognizing the migratory neural crest cell; and S-100, a protein expressed by sustentacular cells of the adrenal medulla. HNK-1 immunoreactivity was found in both chromaffin and nonchromaffin cell types at different times during development, marking the nonchromaffin lineage during the second trimester of gestation as well as the chromaffin lineage in the neonatal period. In addition, S-100 expression was noted in some nonchromaffin neuroblasts, and sustentacular cells were first identified at approximately 28 weeks of gestational age. These data suggest a model of human adrenal medullary histogenesis that incorporates the chromaffin, ganglionic, and sustentacular lineages known to constitute the adult adrenal medulla.

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Neural crest derived precursors of the sympathoadrenal cell lineage give rise to two major cell types that differ in a number of morphological, ultrastructural, and biochemical characteristics: principal sympathetic neurons and chromaffin cells of the adrenal medulla. The present article reviews experimental studies performed on cultured adrenal medullary cells and designed to unravel the nature of epigenetic signals governing the developmental choice between the endocrine chromaffin and the neuronal sympathetic phenotype. Emphasis is placed on the role of glucocorticoids in initiation, development, and maintenance of the endocrine chromaffin phenotype and apparently antagonistic influences exerted by nerve growth factor (NGF) in vitro, resulting in the acquisition of neuronal properties by differentiated chromaffin cells. Experimental data from in vitro studies are compatible with the following conclusions. Glucocorticoids represent the decisive signal for the initial induction of endocrine differentiation. Moreover, high steroid hormone concentrations, as present in the adrenal medulla, are a prerequisite for the maturation of chromaffin cells. Even in a differentiated state, the endocrine phenotype is unstable in the absence of glucocorticoids, and the cells seem to reenter the neuronal developmental pathway. Under these conditions, cellular survival and differentiation into sympathetic neurons become NGF-dependent, as in normal sympathetic development. Thus, the effects of NGF survival, neurite outgrowth, and transmitter synthesis of cultured chromaffin cells probably do not reflect the induction of a specific phenotype, but they may be interpreted as a general neurotrophic support observable with other responsive cell types.

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An attempt has been made to determine the relative functional activities of adrenal and extra-adrenal chromaffin tissue during the neonatal phase and up to 1 week of age using young rabbits and guinea-pigs whose main extra-adrenal abdominal para-aortic chromaffin bodies are respectively non-innervated and innervated. Amine synthesis and storage were followed by assay and autoradiography after a single intraperitoneal injection of L-[2,5,6-3H]DOPA and the findings correlated with amine content as assessed by high performance liquid chromatography and by volume of tissue. The results indicate that in the guinea-pig, in spite of differences in proportions of adrenaline and noradrenaline in the adrenal gland and para-aortic body (PAB), the loss of labelled catecholamines from the innervated PAB closely follows that from the adrenal medulla, suggesting that both participate in normal sympathoadrenal activity. By comparison, in the rabbit the PAB shows only a minimal decreased labelled amine during the first week of life during which period the amine content and concentration of the PAB doubles: the functional significance of this non-innervated extra-adrenal chromaffin tissue, which persists throughout life, has still to be determined.

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Addition of nerve growth factor to cultures of dissociated rat adrenal medullary cells caused fiber outgrowth from chromaffin cells. These fibers exhibited all the characteristics of neurites, particularly the formation of typical growth cones exhibiting intense catecholamine-specific fluorescence. Because this nerve growth factor-mediated neurite outgrowth could be abolished by physiological concentrations of glucocorticoids, it is concluded that the high glucocorticoid concentrations normally present in the adrenal medulla prevent the fiber outgrowth from medullary chromaffin cells in vivo. In dissociated sympathetic neurons the same concentrations of glucocorticoids markedly reduce but do not completely abolish neuronal fiber outgrowth.

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The role of post-synaptic cells in the development of pre-synaptic cholinergic nerve terminals has been investigated in immature rat superior cervical ganglia (SCG) and adrenals employing chemical agents which prevent the normal maturation of post-synaptic cells. A marked atrophy of ganglion adrenergic neurons after guanethidine administration was accompanied by the complete failure of normal maturation of choline acetyltransferase (ChAc) activity in the presynaptic endings. However, the same treatment failed to alter the levels of ChAc in the mature ganglia despite the marked atrophy of adrenergic neurons. Administration of dexamethasone resulted in a growth retardation of ganglion neurons as well as adrenal chromaffin cells reflected by the lower levels of tyrosine hydroxylase and dopamine-beta-hydroxylase than those in untreated tissues. The levels of ChAc were significantly lower in the ganglia, but not in the adrenals when treatment was started immediately after birth. These results support the view that the normal synapse formation in the SCG depends on the normal maturation of adrenergic neurons, and suggest that this dependence is detectable only during a limited period of life.

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Ganglion cells and paraganglionic (PG) cells in the developing rat superior cervical ganglion were studied following postnatal treatment with p-chlorophenylalanine (pCPA) for 5 to 8 days. Litter mates, injected with saline solution, served as controls. Ganglion cells of control animals were differentiated ultrastructurally according to L. Eränkö (1972a) into late sympathicoblasts and young sympathetic nerve cells. In both maturation stages treatment with pCPA caused marked swelling of mitochondria, concomitant with minor changes of other cell organelles. Parallel to the ultrastructural alterations, fluorescence microscopy and cytophotometry revealed a slight diminution of diffuse fluorescence intensity in sympathetic neurons as the expression of a mainly extragranular amine depletion. In distinction from ganglion blocking agents the alterations are regarded as a general toxic effect of pCPA upon maturing sympathetic neurons, which secondarily influences catecholamine storage sites. Following treatment with pCPA, in PG-cells an alteration of mitochondria was scarcely to recognize. Specific granules were distinctly decreased in number, in some cases to an almost complete degree. Concordant to ultrastructural observations a marked diminution of fluorescence intensity was demonstrable in SIF-cells. In addition in these elements the fluorescence spectrum shifted towards the green field. Fluorescence cytophotometric evaluations confirmed the optical impression. Provided, that PG-cells, demonstrated with electron microscopy, are identical with SIF-cells in fluorescence microscopy, the results are discussed on the basis of a specific decrease of primary catecholamines due to an enzyme inhibition involved in catecholamine synthesis.

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