Nerve growth factor collaborates with myocyte‐derived factors to promote development of presynaptic sites in cultured sympathetic neurons

Nerve growth factor (NGF) acutely modulates synaptic transmission between sympathetic neurons and their cardiac myocyte targets. NGF also has developmental effects in establishing the level of synaptic transmission between sympathetic neurons and myocytes in culture, although little is known about the mechanisms by which NGF influences this synaptic connectivity. Here we report that NGF acts in conjunction with factors produced by cardiac myocytes to promote neuronal contact with the target and the extension of synaptic vesicle‐containing growth cones. In conjunction with previously published results showing that NGF has long‐term effects on synaptic transmission between sympathetic neurons and myocytes, this work suggests that NGF acts to promote sympathetic neurotransmission by increasing the number of sympathetic fibers establishing target contact. Further, we found that developmental changes in cardiac myocytes led to an increase in the density of synaptic vesicle–containing variocosities along sympathetic fibers, a process regulated by NGF. Thus, as myocytes mature they produce factors that promote the formation of sympathetic presynaptic structures. These results argue that multiple target interactions regulate the extent of synapse formation between sympathetic neurons and cardiac cells and suggest that NGF promotes presynaptic development by increasing neuronal contact with myocyte‐derived cell surface or matrix‐associated factors. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 460–476, 2000     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

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.     

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.     

Ultrastructural studies on the intracellular fate of 125I-nerve growth factor in cultured rat sympathetic neurons

Primary cell cultures of sympathetic neurons from rat were exposed to 125I-nerve growth factor (NGF) and the fate of the NGF in the cell was followed using electron microscopic autoradiography. The intracellular localization of NGF was determined in the cell bodies and in the proximal neurites of neurons that had been grown in three-chamber dishes, following 5 or 24 hr of retrograde transport of NGF from the distal portions of the neurites. Label in the proximal neurites was predominantly associated with lysosomes and multivesicular bodies (MVBs), and at 5 hr elongated tubular elements were especially heavily labeled. Most of the label in the cell bodies was concentrated in lysosomes and MVBs. Lysosomes accounted for the largest fraction (45-60%) of the grains in the cell body, with a labeling density (LD = % grains/% area) of 3-5, while MVBs accounted for 5-10% of the grains with an LD of 5-20. We observed no evidence of nuclear labeling after 5 or 24 hr of retrograde transport. Mass cultures of neurons were incubated for 22 hr with NGF in the presence of the lysosomal inhibitors chloroquine (CQ, 0.05 mM) or methylamine (MA, 10 mM). In both agents the lysosomes were swollen with membranous material but still sequestered NGF, especially in CQ where the lysosomes were associated with almost 65% of the grains and had an LD of 6. CQ and MA had different effects on the morphology of the MVBs: in CQ they were few in number and compact while in MA they were numerous and appeared swollen and vacuolated. We observed no evidence for the nuclear accumulation of NGF even in the presence of the lysosomotropic agents.     

Caffeine promotes survival of cultured sympathetic neurons deprived of nerve growth factor through a cAMP-dependent mechanism.

The effects of caffeine on neuronal survival independent of trophic factor support were examined in developing superior cervical ganglion in vitro. We found that caffeine promoted neuronal survival in the absence of nerve growth-factor (NGF) in a dose-dependent manner (EC50 = 6 mM). Pulse treatment with caffeine or high K+ (40 mM), which caused only a transient increase in intracellular free Ca2+ levels ([Ca2+]i), did not promote survival. In contrast, caffeine potentiated the saving effect of various phosphodiesterase inhibitors including theophylline (EC50 = 3 mM) and 3-isobutyl-1-methylxanthine (EC50 = 0.4 mM). Non-xanthine phosphodiesterase inhibitor Ro 20-1724 potentiated the survival promoting effect of caffeine or IBMX. Indeed, administration of 20 mM caffeine rapidly restored the cAMP level of NGF-deprived neurons to normal (0.34 pmol/well) within 10 min; the level reached a plateau level (0.69 pmol/well) at 10 h. Even after 1 day, the sustained level was maintained in the presence of caffeine. In contrast, noradrenaline and isoproterenol, which cause only a transient increase in cAMP levels, did not support survival. These data, in conjunction with others, suggest that sustained levels of second messengers, including not only the [Ca2+]i but also the cAMP level, would support the survival of superior cervical ganglion cells independent of trophic factor support.     

Nerve growth factor regulates central terminals of primary sensory neurons

Transection of peripheral sensory axons results in transganglionic degenerative atrophy of central terminals of the affected primary sensory neurons. Nerve growth factor applied at the central stump of the transected nerve prevents or delays transganglionic degenerative atrophy. It is concluded that, under normal conditions, nerve growth factor taken up by receptors at peripheral sensory nerve endings and transported retrogradely to perikarya in dorsal root ganglia, regulates synthesis of neuroproteins destined for maintenance of central terminals of these neurons. Accordingly, transganglionic degenerative atrophy is the consequence of failure of nerve growth factor to reach perikarya of primary sensory neurons.     

Early presynaptic changes during plasticity in cultured hippocampal neurons

Long-lasting increase in synaptic strength is thought to underlie learning. An explosion of data has characterized changes in postsynaptic (pstS) AMPA receptor cycling during potentiation. However, changes occurring within the presynaptic (prS) terminal remain largely unknown. We show that appearance of new release sites during potentiation between cultured hippocampal neurons is due to (a) conversion of nonrecycling sites to recycling sites, (b) formation of new releasing sites from areas containing diffuse staining for the prS marker Vesicle-Associated Membrane Protein-2 and (c) budding of new recycling sites from previously existing recycling sites. In addition, potentiation is accompanied by a release probability increase in pre-existing boutons depending upon their individual probability. These prS changes precede and regulate fluorescence increase for pstS GFP-tagged-AMPA-receptor subunit GluR1. These results suggest that potentiation involves early changes in the prS terminal including remodeling and release probability increase of pre-existing synapses.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.     

Nerve growth factor, sphingomyelins, and sensitization in sensory neurons

Because nerve growth factor(NGF)is elevated during inflammation,plays a causal role in the initiation of hyperalgesia     

Nerve growth factor and fibroblast growth factor influence post-fusion expression of Na-channels in cultured rat skeletal muscle

We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)-sensitive Na-channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H]saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the physiological expression of Na-channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose-related increases in Na-channels compared with myotubes maintained in normal, serum-supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potential. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX-binding induced by GF treatment. The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.     

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.     

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.