Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Developmental studies

Adrenergic sympathetic neurons were grown for 4 wk in submaximal and saturating concentrations of nerve growth factor (NGF) in the virtual absence of non-neuronal cells. In 0.2 or 5 microgram/ml 7S NGF, the neurons gradually decreased in number during the first week, although fewer neurons died at the higher level. No significant change in cell number was observed thereafter. Total neuronal protein, a measure of cell growth, increased linearly with age in both concentrations of NGF. At each age, neurons in high NGF exhibited greater growth per cell than those in low NGF. The ability of neurons to produce catecholamine (CA) increased dramatically during the second and third weeks in both concentrations of NGF, and along a similar time-course, although neurons in submaximal NGF developed a lesser capacity for CA production. As neurons developed in culture, they became less dependent on NGF for survival and CA production, but even in older cultures, approximately 50% of the neurons died when NGF was withdrawn.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.     

The thoracic sympathetic neurons of the chick: normal development and the effects of nerve growth factor

The generation and degeneration of sympathetic neurons in the third thoracic ganglion (segment 19) of the chick were studied between embryonic days (E) 7-18 using 3H-Thymidine autoradiography and routine cell counts. Cumulative radiolabelling experiments indicated that few sympathetic neurons were generated on E6-7. 10% of the sympathetic neurons were generated on E8 and a further 20% on E9. The final 70% of neurons completed the mitotic cycle between E10-12. Cell counts demonstrated that the neuronal population increased from 10,166 +/- 423 (mean +/- SEM) to 22,291 +/- 767 between E8-10 and remained stable up to E14. The population subsequently declined by 37%, to 14,157 +/- 831, by E18. Pyknotic neurons were found at all stages of development, but were most apparent between E7-15. The effects of Nerve Growth Factor (NGF) on the number of both surviving and pyknotic neurons in the ganglion were also examined. E9 embryos treated with NGF from E5-8 showed a 57% increase in the number of sympathetic neurons. This increase therefore occurred prior to the decline in neuronal number and was not accompanied by a decrease in the number of visibly pyknotic neurons. It is therefore possible that early NGF treatment increases the number of sympathetic neurons through a mechanism other than the attenuation of cell death.     

Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: correlation with survival factor requirements in culture

The presence of nerve growth factor receptors and the imipramine-sensitive uptake of catecholamines in sympathetic neurons of chick embryos were investigated by autoradiography. Neurons were dissociated from paravertebral ganglia of different embryonic ages and receptors and catecholamine uptake were then determined both at the beginning of culture and after 2 days in culture, at which time the number of surviving neurons is determined by the survival factors present. It was found that while essentially all the neurons specifically bound 125I-NGF both after dissociation and at the end of the culture period, only 60% of the neurons take up [3H]norepinephrine after dissociation, and this proportion remained constant through the culture period under conditions where all the neurons survived. All of the neurons maintained by NGF in culture (35% of the total) displayed this uptake, and in contrast, only one-quarter of those maintained by heart cell-conditioned medium alone (60% of the total) took up catecholamines. The uptake was shown to be neither induced by NGF nor suppressed by heart cell-conditioned medium. These results support the hypothesis that chick sympathetic ganglia contain discrete subpopulations of neurons which may be selected in culture by virtue of their different requirements for survival factors.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (～2 × 10^?11M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.     

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

Merkel cells in vitro: production of nerve growth factor and selective interactions with sensory neurons

A method has been developed for obtaining mixed primary cultures of dissociated epidermis enriched in Merkel cells. Merkel cells obtained from embryonic rat buccal pads were grown in serum-free medium and identified in vitro using a variety of histological and immunohistochemical markers. Quinacrine, a fluorescent amine, which has been used to identify Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. Cells labeled with quinacrine had a large, phase bright nucleus with prominent nucleoli, surrounded by a phase dark perinuclear ring. Antibodies directed against neuron-specific enolase, another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of quinacrine fluorescent cells. Electron microscopic examination of our cultures indicated that cells containing characteristic features of Merkel cells including cytoplasmic dense-cored granules were present. A small but significant increase in the number of Merkel cells was observed over time in culture. Merkel cells supported the survival and outgrowth of both trigeminal ganglion sensory neurons and sympathetic neurons from the superior cervical ganglion in serum-free medium in the absence of exogenous nerve growth factor (NGF). Immunoblots probed with antibodies directed against NGF demonstrated that NGF was present in the medium taken from these cultures. NGF-like immunoreactivity colocalized to cells containing quinacrine fluorescence in situ and in vitro. Addition of antibodies directed against NGF to cocultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and decreased survival of sensory neurons by 60%. These results suggest that Merkel cells are capable of providing trophic support for their normal complement of sensory neurons by producing NGF. Selective recognition of these targets was studied in vitro by characterizing the interactions between Merkel cells and growth cones from sensory or sympathetic neurons using both time-lapse videomicroscopy and standard morphometry of fixed cocultures. The majority of trigeminal ganglion sensory neurons (approximately 60%) extended growth cones onto clusters of Merkel cells. Neurites which contacted clusters of Merkel cells were significantly more highly branched than those growing on collagen. In contrast, the majority of sympathetic neurons (greater than 90%) failed to grow onto Merkel cells. Growth cones of sympathetic neurons often "collapsed" and retracted when contact was made with a cluster of Merkel cells. Fixation of Merkel cells with paraformaldehyde prior to coculture did not affect this difference between sensory and sympathetic neurite extension onto the Merkel cells. However, prior fixation of Merkel cells eradicated the apparent Merkel ce-induced branching of sensory neurites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of nerve growth factor in plasticity of forebrain cholinergic neurons

Neuronal plastic rearrangements during the development and functioning of neurons are largely regulated by trophic factors, including nerve growth factor (NGF). NGF is also involved in the pathogenesis of Alzheimer’s disease. In the brain, NGF is produced in structures innervated by basal forebrain cholinergic neurons and retrogradely transported along the axons to the bodies of cholinergic neurons. NGF is essential for normal development and functioning of the basal forebrain; it affects formation of the dendritic tree and modulates the activities of choline acetyltransferase and acetylcholinesterase in basal forebrain neurons. The trophic effect of NGF is mediated through its interactions with TrkA and p75 receptors. Experimental and clinical studies have shown that brain levels of NGF are altered in various pathologies. However, the therapeutic use of NGF is limited by its poor ability to penetrate the blood–brain barrier, adverse side effects that are due to the pleiotropic action of this factor, and the possibility of immune response to NGF. For this reason, the development of gene therapy methods for treating NGF deficit-associated pathologies is of particular interest. Another approach is creation of low molecular weight NGF mimetics that would interact with the corresponding receptors and display high biological activity but be free of the unfavorable effects of NGF.     

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 micrograms/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (greater than or equal to 90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (greater than 80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 +/- 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 +/- 13 micron after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 micrograms/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deprivation of nerve growth factor rapidly increases purine efflux from cultured sympathetic neurons

The efflux of [3H]purines from cultured sympathetic neurons prelabelled with [3H]adenine is accelerated 2-3-fold within hours of nerve growth factor (NGF) withdrawal and is reduced by readdition of NGF. Addition of 8-(4-chlorphenyl-thio) cAMP, which delays neurite degeneration, reduced the enhanced efflux of purines, as did the addition of cycloheximide, MgCl2 and the protease inhibitor tosyl-L-lysine chloromethyl ketone. Colchicine accelerated purine efflux and neurite degeneration but 2-deoxyglucose increased purine efflux without inducing degeneration, suggesting that ATP reduction itself is not the cause of neurite degeneration. The increase in purine efflux is thus an early biochemical event that has diagnostic value for the study of NGF action since deprivation is detected well before irreversible changes become established.     

Metabolic requirements for differentiation of embryonic sympathetic ganglia cultured in the absence of exogenous nerve growth factor.

The embryonic mouse superior cervical ganglion (SCG) in culture was employed to define the role of ongoing metabolic processes in morphological and biochemical development. The 14 gestational day SCG does not require added nerve growth factor (NGF) for differentiation in vitro. Consequently, its use allows study of intraganglionic regulation of neuronal growth in the absence of complicating, exogenous growth factors. Ganglia were cultured without added NGF, in medium containing various metabolic inhibitors; neurite elaboration and development of tyrosine hydroxylase (T-OH) activity, a biochemical marker of adrenergic maturation, were evaluated. Neurite elaboration proceeded normally with inhibition of RNA synthesis by actinomycin D, or of protein synthesis by cycloheximide or puromycin. In contrast, inhibition of RNA or protein synthesis prevented normal development of T-OH activity. However, neurites and T-OH developed normally in the presence of DNA synthesis inhibition by cytosine arabinoside, which markedly reduced the nonneuronal cell population. These observations suggest that neurite elaboration and the ontogenetic increase in T-OH activity are regulated differently in ganglia cultured in the absence of exogenous NGF. Moreover, the initial outgrowth of neurites and increase in T-OH activity may occur independent of peripherally migrating support cells in this embryonic ganglion.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons

Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (～2 × 10^?11M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.     

Transforming growth factor beta has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor.

Transforming growth factor beta (TGF beta) influences the growth and differentiation of a wide variety of nonneuronal cells (nnc) during embryogenesis and in response to wounding. In the present study TGF beta 1 and TGF beta 2 were examined for their neurotrophic actions on neonatal rat dorsal root ganglion (DRG) neurons with ganglionic nnc in dissociated cultures. TGF beta 1 and TGF beta 2 each increased both neuronal survival and levels of the peptide neurotransmitter substance P (SP) expressed per neuron as well as per culture. TGF beta 1 was maximally effective at a concentration of 40 pM, whereas TGF beta 2 was about 10-fold less potent. Survival effects promoted by simultaneous treatment with both factors were not additive. TGF beta 1 also changed the morphology and distribution of DRG nnc which resulted in clustering of DRG neurons on top of the nnc. Cotreatment of the cultures with two different anti-nerve growth factor (NGF) antibodies eliminated the neurotrophic effects of TGF beta 1. However, treatment with TGF beta 1 did not alter NGF mRNA expression in the cultures nor did it change the amount of NGF in the medium. Further, TGF beta 1 greatly enhanced survival effects and SP stimulation promoted by exogenous NGF at concentrations up to 100 ng/ml. The neurotrophic effects of TGF beta 1 were significantly attenuated by decreasing the proportion of the ganglionic nnc, suggesting a role for these cells in mediating TGF beta 1 action on the neurons. It is hypothesized that the neurotrophic activity of TGF beta depended upon the presence of molecules immunologically related to NGF and that the effects of TGF beta were synergistic with NGF. These observations suggest that TGF beta may play a role in the differentiation and regeneration of DRG neurons in vivo.     

A Mr 70,000 phosphoprotein of sympathetic neurons regulated by nerve growth factor and by depolarization

The effect of nerve growth factor (NGF) on protein phosphorylation was investigated in cultures of dissociated, purified chick sympathetic neurons labeled with inorganic [32P]phosphate or [35S]methionine. For at least 90 min after dissociation and purification of the neurons, overall protein phosphorylation was similar in the absence and presence of added NGF, indicating that the neurons were not unspecifically affected by this period of NGF deprivation. Addition of NGF resulted in a marked decrease in the phosphorylation of a 70,000-dalton protein, designated p70 . p70 existed in five isoelectric variants, referred to as p70 /1-5. p70 /1 was unphosphorylated and was the least acidic variant. p70 /2-5 contained progressively more phosphate and they were increasingly acidic. NGF, via dephosphorylation (or via a highly specific and very limited proteolysis), induced the conversion of p70 /5 and p70 /4 to p70 /2. The effect of NGF on p70 involved phosphothreonine residues to a greater extent than phosphoserine residues and occurred rapidly, being detectable after 5 min and complete after 15 min. 8-Br-cAMP did not mimic the effect of NGF on p70 . Depolarization of the neurons with high K+ and addition of the calcium ionophore A23187 produced effects on the phosphorylated p70 variants similar to those induced by NGF. The response of p70 to two distinct "survival factors," NGF and depolarization, may suggest a role for this phosphoprotein in the survival of sympathetic neurons.     

Reduction in nerve growth factor availability leads to a conditioning lesion-like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.     

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 micron from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the tau microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 micron), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30-100 micron). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.