Search

Fibroblast growth factor-2 (FGF-2; basic FGF) is widely distributed in the developing and adult brain and has numerous effects on cultured and lesioned neural cells. The physiological role of FGF-2 in the unlesioned nervous system, however, is still not understood. We have studied the distribution of FGF-2 in the developing, adult, and functionally impaired central auditory system of the rat using specific antibodies and peroxidase-antiperoxidase immunocytochemistry. FGF-2-like immunoreactivity (FGF-2-IR) occurred in neuronal cell bodies and/or nerve fibers but was very rarely observed in glial cells. Several auditory brainstem nuclei, including the superior paraolivary nucleus, the medial superior olive, the lateral and ventral trapezoid nuclei, and the central nucleus, as well as the external cortex of the inferior colliculus, were entirely devoid of FGF-2-IR. In the dorsal cochlear nucleus, the lateral superior olive, and the nuclei of the lateral lemniscus, FGF-2-IR was not detectable in nerve cell bodies prior to adult age. Neurons in the medial geniculate body exhibited FGF-2-IR only transiently, from postnatal day (P) 5 until P16. Neurons in the medial nucleus of the trapezoid body were immunoreactive from P8 onwards. FGF-2-IR in anteroventral and posteroventral cochlear neurons disappeared at P14, i. e., at the onset of hearing, but immunoreactivity returned after P21. A transient expression of FGF-2 around the time when hearing function commences was observed in the dorsal cortex of the inferior colliculus. Thus, regulation of neuronal FGF-2-IR in several, but not all, auditory, nuclei is related to the onset of hearing, in that IR disappears at that time or transiently appears. This suggests a causal link between the onset of hearing and FGF-2 expression. In support of this notion, ototoxic treatment with gentamycin abolished FGF-2-IR in the P16 medial geniculate body but not in other auditory brainstem centers. Thus, FGF-2 may be considered a regulator or indicator of the acquisition of functional activity and responsiveness to sensory stimuli in several areas of the auditory system. © 1995 Wiley-Liss, Inc.

The sympathoadrenal cell lineage originates from the neural crest and comprises the neurons of sympathetic ganglia, adrenal and extra-adrenal chromaffin cells, and the so-called small intensely fluorescent cells. In vitro studies using mammalian immature chromaffin cells, adrenal or sympathetic ganglionic progenitors, or ganglionic small intensely fluorescent cells, have suggested that glucocorticoid hormones are essential for inhibiting neuronal differentiation of sympathoadrenal progenitors and promoting the chromaffin cell phenotype. In avian systems, however, the distinct cellular phenotypes in this lineage and the molecular cues underlying their differentiation have not been fully explored. In the chick embryo, early sympathetic ganglion anlagen are populated by granule-containing cells that morphologically resemble small intensely fluorescent cells and chromaffin cell progenitors. These cells subsequently disappear from the ganglia, by death and by transition into fully differentiated sympathetic neurons, as indicated by the appearance of cells that are ultrastructurally intermediate between granule-containing cells and fully differentiated neurons (granule-containing cells in transition). In the present study, we show that treatment of cultured sympathetic cells dissociated from embryonic day (E) 7, 9, or 11 lumbar sympathetic ganglia with the glucocorticoid hormones hydrocortisone or corticosterone has neither an inhibitory nor an inductive effect on phenotypes of granule-containing cells or granule-containing cells in transition. In cell cultures of E15 ganglia, however, glucocorticoid treatment induces a granule-containing cell resembling the granule-containing phenotype. These results suggest that the early granule-containing cells and granule-containing cells in transition in chick sympathetic ganglia are not the counterparts of glucocorticoid-responsive mammalian small intensely fluorescent or chromaffin progenitor cells, despite their morphological similarity. However, E15 sympathetic ganglia apparently contain a glucocorticoid-responsive progenitor population that can differentiate into chromaffin-like cells. These progenitors seem to require a systemic or intraganglionic developmental signal or undergo a temporal switch that renders them susceptible to glucocorticoids.

A recently cloned neurotrophic factor, Glial Cell Line-Derived Neurotrophic Factor (GDNF), has been implicated in the survival and morphological and functional differentiation of midbrain dopaminergic neurones in vitro. GDNF has therefore been proposed as a factor which may have utility in the treatment of Parkinson's disease. In the present study, we have used RT-PCR to analyse the distribution of GDNF mRNA throughout the newborn rat (P0). We show that GDNF transcripts are present in kidney, lung, bone, heart, liver, spleen, sciatic nerve and blood. Two separate GDNF transcripts are present in different ratios in each tissue investigated. Sequence analysis of both these mRNA species revealed that the shorter transcript (sGDNF) contains a deletion of 78 bp in comparison to the published sequence for GDNF. We speculate that this shorter mRNA arose due to alternative splicing.

Transforming growth factors beta are multifunctional proteins and regulators of cell proliferation and differentiation. Transforming growth factor-beta s have the capacity to rescue adult neurons from ischemia- and glutamate-induced cell death and are prominent in the embryonic and adult brain including striatum and substantia nigra. In the present study we show that transforming growth factors-beta 1, -2, and -3 promote, in a dose-dependent fashion, in vitro survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the embryonic rat mesencephalon floor. The magnitude of the effect, which was half-maximal at a concentration of 20 pM, was identical for all three transforming growth factor-isoforms and matched that of fibroblast growth factor-2. Unlike fibroblast growth factor-2, however, transforming growth factor-beta s did not increase numbers of astroglial cells visualized by using antibodies to glial fibrillary acidic protein, and had no effect on cell proliferation monitored by incorporation of BrdUrd. Transforming growth factor-beta s were significantly more potent than fibroblast growth factor-2 in protecting dopaminergic neurons against N-methyl-4-phenylpyridinium ion toxicity. RT-PCR analysis indicated that the effect of transforming growth factor-beta s is not mediated by glial cell-derived neurotrophic factor, which was not detectable in cultures at various time points. On the other hand transforming growth factor-beta 2 mRNA could be detected in freshly isolated and cultured mesencephalic cells, and its immunoreactivity has also been demonstrated in the embryonic day 14 mesencephalon floor. We conclude that transforming growth factor-beta has trophic and protective effects on developing dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of CNTF mRNA was investigated in primary cortical astrocytes cultured from newborn rats and in C6 glioma cells. Northern blot analysis indicated that semi-confluent astrocyte cell cultures. CNTF added to confluent astrocyte cultures down-regulated CNTF message in a dose-dependent fashion. In contrast, when CNTF was given to semi-confluent astrocyte cultures the level of CNTF mRNA was up-regulated. C6 glioma cells also showed a cell density-dependent expression of CNTF: cells from confluent cultures expressed detectable amounts of CNTF mRNA whereas those from semi-confluent cultures did not. Thus, CNTF mRNA expression by astroglial and glioma cells is regulated by cell contact and exogenous CNTF in vitro.

Summary We have studied in vitro the effects of HOE-BAY 946, a specific inhibitor of tyrosine kinase C, on spontaneous and basic FGF-induced proliferation of the epithelial cell lines A549 and HELA as well as the neuroblastoma cell line SY5Y using a fully automated colorimetric assay for determinations of cell numbers. HOE-BAY suppressed spontaneous proliferation of the epithelial lines, but not the neuroblastoma line, both in the presence and absence of serum. In contrast, the drug completely abolished the mitogenic effect of basic FGF on all cell lines. This suggests an involvement of a tyrosine kinase C activity in the transduction of the FGF signal both in the epithelial and neuroblastoma lines. Since the action of FGF on non-transformed neuronal cells is accompanied by a down-regulation of kinase C, and kinase C activation blocks the FGF effects, our data indicate that differentiative and mitogenic effects of FGF on neuronal cells may employ different transduction mechanisms.

The transforming growth factors-beta (TGFs-beta) are multifunctional peptide growth factors that have been localized in neuronal and glial cells of the CNS of mice, rats, and chick embryos. We tested the TGF-beta isoforms 1, 2, and 3 for their protective effects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid L-glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding 1 mM sodium cyanide to the culture medium for a period of 30 min. Treatment with TGF-beta 1 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels, and protein content of the cultures assessed 72 h after the toxic insult. TGF-beta 3 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ml, whereas TGF-beta 2 only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and post-treatment with TGF-beta 1 also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to 1 mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-beta 1 (0.3-3 ng/ml), and to a lesser extent TGF-beta 3 (0.1-1 ng/ml), significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 mM L-glutamate. These results demonstrate that the TGFs-beta are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.

The protective role of basic fibroblast growth factor (FGF-2) for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and methylpyridiniumion (MPP+)-lesioned dopaminergic (DAergic) nigrostriatal neurons was studied, using dissociated cell cultures of embryonic day (E) 14 rat mesencephalon. Cells were grown in different culture media and received FGF-2 (5 ng/ml) and/or the toxins (5 μM) at various schedules, but were consistently allowed to differentiate for 3 days prior to becoming exposed to the toxin. Survival of tyrosine hydroxylase (TH)-immunoreactive cells at 7 days was only markedly impaired by MPTP, if horse serum (HS) or bovine serum albumine (BSA) were omitted from the culture medium. FGF-2 increased the number of TH-immunoreactive cells, and this increase not diminished by MPTP under any culture condition. Uptake of 3H-DA was significantly reduced by MPTP in HS- and BSA-containing, but not in protein-less cultures. A protective effect by FGF-2 was only seen in the presence of BSA. MPP+ caused a more pronounced reduction in 3H-DA uptake than MPTP, and this effect was partially reversed by the addition of FGF-2, unless cultures contained HS. Neurofilament protein (NF), an indirect measure for the total number of neurons present in the cultures, was not significantly reduced by MPTP or MPP+ corroborating the specificity of the toxin for DAergic neurons, which constitute only a minor fraction in these cultures. In line with the wide spectrum of target neurons of FGF-2, this factor significantly increased NF contents under any culture condition. Quantification of the amounts of glial fibrillary acidic protein (GFAP) revealed stimulatory effects of FGF-2 (2.5- to 4-fold) and at least 10-fold higher levels in the presence as compared to the absence of HS. These data show that FGF-2 can protect DAergic neurons against MPTP- and MPP+-mediated damage. However, the effects of the toxins as well as of FGF-2 are partially dependent on culture conditions. Variations in the effectiveness of toxins and FGF-2 are not overtly related to the total numbers of neurons or astroglial cells, but may reflect culture type-dependent alterations of neuronal and glial metabolism. © 1993 Wiley-Liss, Inc.

The multifunctional proteins, basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF), share a capacity to promote in vitro and in vivo survival of several, partly overlapping neuron populations. Whether they can affect neurons directly or whether their supportive effects are mediated by non-neuronal cells and their growth factor products has been addressed in this study by establishing single neuron cultures from embryonic chick ciliary ganglia. Cultures with one or two neurons and without any non-neuronal cells were obtained by limiting dilution of ganglionic cell suspensions on 96-well microtiter plates. In the presence of bFGF about 80% of the wells that contained 1 or 2 neurons at the time of seeding, had this (these) neuron(s) maintained after 1 and 5 days. Absence of bFGF resulted in the death of neurons in over 80% of the wells screened. Identical results were obtained with CNTF. These data demonstrate the effectiveness of bFGF and CNTF at the single neuron level, but do not rule out that the factors may act indirectly on neurons, particularly in complex in vitro and in vivo situations.

We have studied the effects of basic fibroblast growth factor (bFGF), which occurs in the adrenal medulla, on the survival, morphological phenotype, storage capacity for catecholamines and induction of the synthesizing enzymes tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) of cultured chromaffin cells from young postnatal rats. Basic FGF (40 ng/ml), like nerve growth factor (NGF; 40 ng/ml) prevented a drastic numerical decrease of chromaffin cells over a 4-day culture period, but, in contrast to NGF, did not induce neurite outgrowth, unless the cells were maintained for 7 days. Basic FGF was also more effective than NGF in maintaining the initial storage capacity for catecholamines, and even increased it under certain culture conditions (laminin instead of polyornithine, or 200 ng instead of 40 ng/ml). Basic FGF and NGF did not induce TH and PNMT activities beyond their initial levels, but partially prevented the reduction of TH activity seen after 4 days in culture. Based on the present data and the previously reported greater in vitro survival and transmitter stability of older chromaffin cells, which contain bFGF, and the relative instability of young postnatal chromaffin cells, which express no or very low levels of bFGF until 8 days postnatally, but respond to it, we hypothesize that bFGF is an important autocrine/paracrine maintenance factor for adult chromaffin cells.

We studied the molecular composition of the neurotrophic/growth factor cocktail synthesized, stored and liberated by adrenomedullary chromaffi cells. Chromaffin cells have a dual capacity as models of the neuronal biochemical machinery and as therapeutic tools (implants) in several neural disorders, most notably Parkinson’s disease. Cultured bovine chromaffin cells release a proteinaceous material into their culture medium that, when applied to a wide range of different embryonic neurons from the CNS and PNS in vitro, promotes their viability and neuritic growth /1-3/. Stimulation of the chromaffin cells with cholinergic agonists or high K + enhances the amounts of the trophic mate

The distribution of basic fibroblast growth factor (bFGF)-immunoreactivity (IR) was studied in rat sensory and autonomic ganglia. In postnatal and adult sympathetic superior cervical ganglia and in adult parasympathetic otic ganglia no bFGF-staining was found. Postnatal and adult neural crest- and placode-derived sensory ganglia displayed intensive bFGF-IR in a neuronal subpopulation. This subpopulation was characterized by use of consecutive sections of adult dorsal root ganglia stained with antibodies against substance P, somatostatin, bombesin, and bFGF. Basic FGF was colocalized with the somatostatin/bombesin subpopulation but not with substance P.

The fibroblast growth factors (FGFs) represent a family of peptide growth factors with relatively high amino acid sequence homologies ranging between 39 and 55% (Table 1; see Baird and Bohlen, 1990, for a review). The first two FGFs characterized, acidic and basic FGF (aFGF and bFGF, respectively), occur in most mesenchymal and neuroectodermal tissues. Five other members of the FGF family were identified as proteins encoded by tumor oncogenes. They possess bFGF-like biological activities in terms of their mitogenic effects. Acidic and basic FGF including some N-terminal truncated forms exert similar biological functions, aFGF being 10–100 times less potent than bFGF.

Basic fibroblast growth factor (bFGF) has recently been isolated from bovine adrenal glands. Immunohistological data revealed its presence in both adrenal cortex and adrenal medulla. Using immuno-electronmicroscopy, we found that in medullary chromaffin cells bFGF-immunoreactivity is localized in the secretory granules. Immunoreactivity also was observed by electronmicroscopy in isolated granules. Western blot analysis revealed the presence of the typical 18-kDa bFGF and additional immunoreactive materials with molecular masses of approximately 24, 30, and 46 kDa in whole bovine adrenal, and in cortex and medulla. Similar results were obtained with proteins from bovine chromaffin granules, with the following two exceptions: the 46-kDa immunoreactivity was found to be highly enriched when compared with medulla or cortex, and the 18-kDa band could be detected with only an antiserum against a synthetic peptide comprising the 24 NH2-terminal amino acids of bFGF, and not with an antiserum against purified bovine pituitary bFGF. All fractions enriched for bFGF-immunoreactivity showed neurotrophic activity for chick ciliary ganglion neurons, which could be blocked by antibodies. These results demonstrate for the first time the localization and occurrence of bFGF in a cellular secretory organelle, and present further evidence for the existence of higher molecular weight immunoreactive forms of bFGF.

The specific mechanisms underlying the restorative effects of adrenal chromaffin grafts in experimental parkinsonism are still obscure. Recent findings indicated an involvement of graft-induced trophic interactions in the course of recovery-related events. Evidence that basic fibroblast growth factor (bFGF), a potent trophic protein for neurons, (1) is present in chromaffin cells (Blottner et al., 1989) and (2) exerts trophic activities on embryonic mesencephalic neurons in vitro (Ferrari et al., 1989) provided the rationale for administering bFGF in gel foam implants unilaterally to the striatum of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned mice. Simultaneous bFGF/MPTP treatment diminished bilaterally the reduction of striatal dopamine (DA) levels observed in cytochrome c/MPTP-treated mice and led to an ipsilateral reappearance of tyrosine hydroxylase (TH)-like immunoreactive fibers, most notably adjacent to the implant, 2 weeks after the surgery. Determinations of TH activities and TH immunoblotting demonstrated that bFGF almost fully reversed the loss of TH activity on either side but restored TH protein more on the ipsilateral than on the contralateral side. Furthermore, differences in dihydroxyphenylacetic acid levels, which were about twice as high on the contralateral side yet still reduced with respect to untreated mice, supported our assumption that the molar TH activity was increased on the untreated side, possibly due to an intrinsic compensatory up- regulation. Delayed administration of bFGF starting 8 d after the MPTP treatment was equally effective with regard to morphological parameters. Our results suggest that bFGF partially prevents the deleterious chemical and morphological consequences of an MPTP-mediated nigrostriatal lesion. Thus, bFGF mimics at least the morphological effects of chromaffin cell grafts to the MPTP-lesioned brain.

The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.

An antibody against basic fibroblast growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30–33 and 46 kDa in inununoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.

Naturally occurring neuronal cell death during development is generally accepted to be regulated in part by neurotrophic factors (NTFs) (Berg 1982; Cunningham 1982; Oppenheim 1985). Beyond neural development NTFs are also involved in the maintenance and regeneration of the peripheral and central nervous system (Levi-Montalcini 1972; Johnson et al. 1980; Gage et a. 1986).

Long-term maintenance of neurons and their target cells is mediated by trophic molecules delivered through anterograde and retrograde axonal flow to the respective destinations. While trophic anterograde mechanisms by which, e.g., a neuron can interfere with the differentiation of its target cells are only beginning to be explored (Anderson and Fambrough 1983; Sames et al. 1984), retrograde trophic effects have been studied in great detail using nerve growth factor (NGF), the best established neurotrophic protein, as a paradigm (Levi-Montalcini 1987).

This study describes the survival and neurite outgrowth behaviors of cultured adrenal medullary (chromaffin) cells obtained from postnatal rats 1 day (D1) to 100 days (D100) old in response to nerve growth factor (NGF), chick eye ciliary neuronotrophic factor (CNTF), and laminin. In the absence of trophic factors the 4-day survival of cultured chromaffin cells (relative to the number of cells attached at 2 hr) increased from one-third of the cells at D1 to 40% at D8 and 90-100% at D16 and older stages. At saturating concentrations NGF increased cell survival at D8 by 90%, but failed to support all chromaffin cells present at 2 hr. In contrast, CNTF supported the survival of all cells at D8. At D1 NGF and CNTF had only a very small effect on survival during the 4-day culture period, although both factors clearly enhanced the numbers of surviving cells after 8 days. Either NGF or CNTF also elicited neurite outgrowth from rat chromaffin cells, which amounted to approximately 15-20% at D1 and D8 and subsequently decreased to about 5-8% at D30 and virtually zero at D100. At this last age both factors applied together clearly elicited neurites. Such a potentiating effect of NGF and CNTF was also seen at earlier postnatal ages. Laminin did not affect neurite growth at D30 in the absence of trophic factors, as already described for D8 rat chromaffin cells. In the presence of NGF, however, it increased neurite length and branching during a 4-day culture period and even enhanced neurite recruitment at later culture times. These data suggest that rat chromaffin cells undergo age-related changes in their responses to NGF and CNTF and that laminin modulates their neurite outgrowth behaviors in the presence of trophic factors.

This study investigates the survival properties and changes in the morphological phenotype of adrenal medullary (chromaffin and neuronal) cells cultured from embryonic chicks at different developmental ages (embryonic days E8 to E16) in response to nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). The 4-day survival of medullary cells from all embryonic ages except E8 was about 80% of the seeded cells and was only slightly enhanced by the addition of saturating doses of CNTF (10 ng/ml). With no factors, after 4 days 10–30% of the surviving medullary cells extended neurites. NGF (100 ng/ml) and, even more, CNTF (10 ng/ml) and their combination substantially increased the proportions of neuritebearing cells (up to 70%). The effect of the factors were maximal at E10 and E12 and declined at older developmental ages. Neurite growth was virtually unaffected by NGF and CNTF at E8. These results show that in vitro survival and neurite growth of chick adrenal medullary cells in response to trophic factors is developmentally regulated.

Using beta-bungarotoxin (beta-BTX) as a tool to eliminate the preganglionic cholinergic nerve supply to the embryonic rat adrenal gland, we have investigated whether or not these nerves affect the differentiation of embryonic chromaffin cells (pheochromoblasts). Rat fetuses received a single injection of 1 or 2 micrograms beta-BTX or an identical volume of saline at embryonic day (E) 17 and were taken for morphological and biochemical analyses at E 21. Administration of beta-BTX caused a 15 to 20% reduction in body weight, crown-rump-length and adrenal weight. Spinal cord development was reduced and acetylcholinesterase-positive cells in ventral and lateral columns were virtually absent in toxin-treated animals. In adrenal glands, a decrease of choline acetyltransferase activity to 13% of control levels and a concomitant decrease of ultrastructurally identifiable nerve fibers and axon terminals revealed that application of 2 micrograms beta-BTX effectively reduced the neuronal input to E 21 adrenal glands. Values for total adrenal catecholamines, relative amounts of adrenaline and noradrenaline, tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities were unaltered. All ultrastructural features of pheochromoblasts (except the lack of synapse-like axon terminals) were inconspicuous. Corticosterone levels in adrenals and plasma were identical to controls. These data strongly suggest that normal embryonic development of adrenal chromaffin cells does not require an intact nerve supply.

Tetanus toxin (TT) and antibodies to neuron-specific enolase (NSE) were used to study the developmentally regulated expression of two neuronal markers in isolated and short-term cultured rat sensory and sympathetic neurons at different postnatal ages. The results show a reciprocal pattern of TT binding and NSE-immunoreactive neurons during postnatal maturation: there is a gradual transition from TT-positive, NSE-negative to TT-negative, NSE-positive phenotypic expressions. We conclude that TT may serve as a neuronal marker for newborn, anti-NSE antibodies as markers for adult neurons, and a combination of both for adolescent neurons.

This introductory article presents a survey of the principle cellular constituents of the sympathoadrenal section in the peripheral autonomic nervous system, their development and plasticity and factors that govern the expression of particular morphologic and transmitter phenotypes. The article focuses on results obtained in cell culture studies with isolated chromaffin cells that have permitted the analysis of molecular signals possibly serving as environmental cues during the development of sympathoadrenal cells.

The presence of fibroblast growth factor (FGF) was investigated by immunocytochemistry in cultured neuronal cells derived from the peripheral (PNS) and the central nervous system (CNS) of chick and rat embryos. Polyclonal antimouse FGF antibodies, which cross-react with basic and acidic FGF, were used in the peroxidase immunocytochemical staining method. FGF immunoreactivity was found in neurons. Staining intensity in chick and rat brain neuronal cells increased during the culture period, reached a maximum after 6-8 days, and subsequently declined. Embryonic chick ciliary and dorsal root ganglionic (DRG) neurons as well as DRG neurons from newborn rat displayed intense FGF immunoreactivity at 12 and 48 hr in culture. Nonneuronal cells were not stained. The data demonstrate for the first time that cultured neurons from the CNS and PNS contain FGF.

Tetanus toxin (TT) binding to cultured rat and bovine adrenal medullary cells has been investigated using indirect immunofluorescence and anti-dopamine-β-hydroxylase (DBH) antibodies as a probe to identify catecholaminergic cells. TT binds to all rat adrenal medullary cells which display a neuronal phenotype induced by treatment with nerve growth factor and/or medium conditioned by C6 glioma cells. In contrast, 90–95% of the rounded DBH-positive cells are TT-negative, suggesting that in vitro-transdifferentiation of rat chromaffin cells alternates the expression of membrane properties. Cultured bovine chromaffin cells have no TT binding sites independent of their morphological phenotype.

Tetanus toxin (TT) was used as a diagnostic marker for human neuroblastoma (NB) cells. TT binding sites visualized by TT and FITC-conjugated anti-TT antibodies were present on NB cells from all 13 cases studied comprising Stages II, III, IV, IVS and histologic grades 1 through 3. NB cells from both bone marrow aspirates and tumor biopsies as well as cultured NB cells were TT-positive. Diagnosis of NB was further ascertained by electron microscopy, cell culture, and quantitative determinations of catecholamines in tumor material. Only electron microscopic diagnoses had an accuracy comparable to that of TT labeling. None of the non-NB tumors (Ewing's sarcoma, acute lymphatic and myeloic leukemia, acute monocyte leukemia, chronic myeloic leukemia, Hodgkin's disease, oat cell carcinoma of the lung, pheochromocytoma), except for the pheochromocytoma, were found to bind TT specifically. These results suggest that TT may be profitably employed as a diagnostic marker of human NB cells. The advantages of the methods are its high discriminative capacity against non-NB cells and rapid applicability.

One subset of neural crest cells, the sympathoadrenal precursors, undergoes a switch in phenotype expression, when they invade the adrenal anlagen and become associated with adrenocortical cells. To investigate the mechanisms responsible for the conversion of noradrenaline synthesizing precursors to adrenaline producing endocrine chromaffin cells we studied the role of glucocorticoids on the initial induction of adrenaline synthesis in embryonic adrenals and cultures of highly purified chromaffin precursor cells. We could show that in vivo differentiation of rat chromaffin precursors commences between 16.3 and 17.3 days of gestation. While adrenaline and the activity of the enzyme phenylethanolamine N-methyltransferase (PNMT), which converts noradrenaline to adrenaline, were present at Embryonic Day 17.3 (E17.3), they were not detectable in E16.3 adrenals. Small amounts of corticosterone were present in E16.3 adrenals and plasma, but in parallel with the initial induction of adrenaline biosynthesis, a sharp rise in organ and plasma glucocorticoid levels occurred until E17.3. Chromaffin precursor cells, isolated at E16.3 and cultured for 4 days, failed to express PNMT activity and adrenaline. However, 0.1 nM dexamethasone was already sufficient for the initial induction of adrenaline and its synthesizing enzyme. Specific glucocorticoid binding of freshly isolated chromaffin (precursor) cells revealed a developmental increase during embryogenesis, yet no glucocorticoid binding sites were detectable in chromaffin precursor cells at E16.3. They appeared at E17.3 in parallel with the initial induction of adrenaline biosynthesis and the enormous rise of adrenal and plasma corticosterone levels. We therefore conclude that glucocorticoids are essential and sufficient to trigger the differentiation of noradrenergic sympathoadrenal precursors to adrenergic chromaffin cells after a functional glucocorticoid receptor system has been established.

Growth cones formed by adrenal chromaffin cells from young postnatal rats cultured in the presence of nerve growth factor (NGF) were studied at an electron microscopic level. These growth cones are similar in many respects to those formed by sympathetic neurons in vitro supporting the view that NGF-treated chromaffin cells may undergo neuronal transdifferentiation.

Adult bovine and young rat chromaffin cells cultured in serum-free medium were examined for their survival and differentiation following exposure to various additives, trophic agents and conditioned media. Adrenal chromaffin cells dissociated from 8 day old rats were maintained by dexamethasone, NGF and CNTF or without any additives in an N1-supplemented medium in similar numbers as in serum-containing medium for up to 6 days. Neuritic growth elicited by NGF or CNTF was enhanced in the absence of serum. Medium conditioned by purified bovine chromaffin cells improved cell survival and caused neurite outgrowth in a dose-dependent manner. The activiti(es) was sensitive to heat and trypsin and not blocked by the addition of anti-NGF antibodies. Bovine chromaffin cell survival was reduced by 30% when cells were maintained for one week in the absence as compared to the presence of serum. Addition of insulin, the N1 supplement, dexamethasone or dbcAMP single or in combinations improved the survival to different extents. A combination of insulin (5 micrograms/ml) and dexamethasone (5 X 10(-6) M) proved to be optimal in this respect. However, these supplements failed to restore the cellular catecholamine, noradrenaline and adrenaline contents to levels seen in the presence of serum. This was also true for a chromaffin cell-conditioned medium, which improved survival without elevating the catecholamine contents. Conditioned medium, however, partly restored a more physiological adrenaline-noradrenaline-ratio.

The effects of medium conditioned by rat C6 glioma cells (C6-CM) on the survival, neurite formation, and catecholamine content of adrenal medullary cells in culture were investigated and compared with the effects of nerve growth factor (NGF). Adrenal medullary cells were isolated from 10-day-old rats and the proportions of surviving and neurite-extending cells were determined after 8 days in culture. In the presence of C6-CM virtually all seeded cells survived and 50% developed neuritic processes. In contrast, NGF did not support survival above control levels (30%) and induced neurite formation from approximately one-third of the surviving cells. C6-CM and NGF had no additive effects on neurite outgrowth. C6-CM-mediated fiber outgrowth was not inhibited by physiological concentrations of glucocorticoids which abolished the NGF-induced neurite formation. Both C6-CM and NGF increased the catecholamine content of the cultures and reduced the relative content of epinephrine. However, in view of its substantial effect on cell survival as compared to NGF, C6-CM caused a reduction of the catecholamine content per cell. We conclude that adrenal medullary cells, like other members of the sensory-sympathetic cell lineage of the neural crest, respond to glial-conditioned medium. This response differs both quantitatively and qualitatively from that mediated by NGF.

Adrenal chromaffin cells from early postnatal rats maintained in culture have previously been shown to grow neuritic processes and survive better in the presence of nerve growth factor (NGF). In the present study we have quantitated the effects on chromaffin cell (postnatal day (D) 8) survival and neurite outgrowth of: NGF, ciliary neuronotrophic factor (CNTF), activities contained in various types of conditioned media (CM), and various substrata (laminin, fibronectin and polyornithine-binding neurite-promoting factor from RN 22 Schwannoma cells - PNPF). At saturating concentrations CNTF (50 ng/ml) and C6 glioma cell CM, (50-fold concentrated) supported survival over the 4-day culture period of all the chromaffin cells present in culture 2 h after seeding. NGF (50 ng/ml) and the non-concentrated CMs from primary Schwann cell and astrocytes as well as Schwannoma and C6 glioma cell cultures, achieved the maintenance of only about half the number of cells above the baseline survival as compared to CNTF and the concentrated C6-CM. These results are compatible with two subsets of D8 chromaffin cells, one only supported by CNTF and the concentrated CM and the other supported by either NGF or CNTF. Either NGF or CNTF elicited neurite outgrowth from 15-20% of the surviving cells. Combination of maximal doses of NGF and CNTF caused a small increase in neurite recruitment beyond that elicited by either factor alone. Low doses of CNTF added to the effect of NGF, shifting the NGF titration curve by about 4-fold. Neurite outgrowth was also induced by the concentrated, but not the unconcentrated C6-CM. Laminin, fibronectin and PNPF did not affect the fibronectin and PNPF did not affect the recruitment of neurites as compared to a polyornithine substratum unless the cultures were supplemented with a neuronotrophic factor and carried for 7 days. However, even before showing effects on neurite recruitment these substrata affected various neuritic performances, such as length, neurite numbers and endings per cell.