Effects of nerve growth factor on autonomic neurons in the chick embryo: A stereological study

Quantitative effects of nerve growth factor (NGF) on the sympathetic, Remak and ciliary ganglia in chicken embryos were investigated. Purified mouse beta NGF was injected (80 micrograms per day for three or four consecutive days) into the yolk sac at different stages (starting on days 6, 8, 10 and 13) of embryonic development. Ganglia were taken for fixation and embedding one day after the last NGF injection. The number of neurons belonging to the different size classes was determined by a computer aided stereological method based on unfolding of cell diameter frequencies. The volume of sympathetic ganglia was increased at all stages with a maximum of 8-fold occurring on day 10. The ganglion of Remak showed a 3-fold volume increase up to embryonic days 10 and 12. Ciliary ganglia did not exhibit any differences in volume or neuron size between the controls and the embryos injected with NGF. The number of neurons was increased in younger sympathetic and Remak ganglia in response to NGF, as was the recruitment of neurons to the larger size classes.     

Comparative analysis of brain neurite-stimulating protein and nerve growth factor by using a culture of chick embryo sensory neurons

The effect of brain neurite-stimulating protein (BNSP) on growth and differentiation of nerve cells was studied in cultured chick embryo neurons BNSP stimulated axon growth. Its chemical characteristics were different from those of neurotrophic factor (NGF). The effect of NGF antiserum was not blocked by the BNSP action. The neurite-stimulating effect of BNSP can be used for stimulation of regenerating processes in the nervous tissue.     

Effects of injecting antibodies to mouse nerve growth factor into the chick embryo

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8- to 15-day-old chick embryo was followed by an enzyme-linked immunoassay. Normal rabbit antibodies (250 μg) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 μg anti-NGF antibodies were injected (40 ng/g). The concentration of anti-NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3–7 were studied in 10-day-old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti-NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti-NGF-treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000–2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti-NGF IgG into the embryo.     

Influence of laminin on the responsiveness of early chick embryo neural tube neurons to nerve growth factor

Dissociated neurons from the neural tube containing the trigeminal motor nucleus from early chick embryos were cultured on laminin or collagen-polyornithine substrates, with and without nerve growth factor (NGF). Control cultures were grown in similar conditions with cytochrome-C. It was found that neuronal survival was not affected by NGF or cytochrome-C, but it was enhanced by laminin. The expression of neuritic processes, however, was significantly enhanced in the presence of NGF on both laminin and collagen-polyornithine surfaces, with the greatest number of neurons producing processes seen in the laminin-plus-NGF group. The length of processes was similarly enhanced by laminin and by NGF. Cytochrome-C did not influence any of these measures. The results indicate that while laminin potentiates the NGF effect on these early neuronal populations, NGF in conjunction with other substrata can have similar, though less dramatic, effects. These results, together with prior evidence of NGF receptors and specific NGF retrograde transport, suggest that this growth factor may play a significant role in the normal ontogeny of early motoneuron populations.     

Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons

We have investigated the effects of nerve growth factor (NGF, 2.5 ng/ml for 1–2 weeks) on enriched adult rat dorsal root ganglion (DRG) neurons maintained in cell culture in defined media. Whole-cell recordings in cells cultured in the absence and presence of NGF revealed no significant difference in resting membrane potential and input resistance. However, the threshold for spike generation was significantly lower in untreated cells than in treated cells; −25 ± 1.1mV vs−19 ± 2.2mV, respectively. The sensitivity of the Na⁺ spike to tetrodotoxin (TTX, 1 μM) was different in cells cultured in the absence or presence of NGF. For example, spikes were abolished by TTX in 100% of untreated cells, while in NGF-treated cells the spike was abolished in only 41% of the neurons. Chemosensitivity of DRG neurons was also different in the absence and presence of NGF. For example, the percent of neurons in which a current activated by 8-methyl-N-vanillyl-6-nonenamide (capsaicin, 500 nM) was detected, increased from 18% in untreated cells to 55% in NGF-treated cells. NGF did not influence the number of cells surviving. The results indicate that NGF can regulate TTX and capsaicin sensitivity in these adult rat sensory neurons. Our experimental protocol indicates that this effect is not mediated by a factor in the serum or released from non-neuronal cells.     

Inhibition of axonal growth from sensory neurons by excess nerve growth factor

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NFG). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.     

Immunohistochemical localisation of nerve growth factor in a subpopulation of chick spinal ganglion neurons

We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Anti-sera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.     

Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons

This study investigated the effects of exogenous nerve growth factor (NGF) on the survival and differentiation in primary culture of sensory neurons isolated from adult (6 months) and aged (2 years) mice. For neurons prepared from adult mice, a concentration effect was evident during a 2 week culture period: Neuronal counts in cultures supplemented with 25 and 50 ng/ml NGF did not differ significantly from those of control cultures without exogenous NGF or those with anti-NGF included in the culture medium, whereas cultures supplemented with either 100 or 200 ng/ml NGF contained higher numbers of neurons throughout the culture period. Cultures prepared from aged mice contained less neurons than those from adult mice, although those supplemented with 100 ng/ml NGF retained higher neuronal numbers than cultures from aged mice which did not receive exogenous NGF. Neuronal diameters were measured to investigate whether specific subpopulations of neurons were more dependent on NGF; the results indicate that neurons of a medium-larger diameter were more prevalent than cells with a smaller diameter following NGF administration. A shape index was calculated for each culture regimen; with longer culture periods a higher proportion of spindle-shaped neurons was observed. © 1993 Wiley-Liss, Inc.     

Different effects of nerve growth factor on cultured sympathetic and sensory neurons

Long-term cultures of dissociated nodose ganglion (NG) and superior cervical ganglion (SCG) neurons from newborn rabbits were used to compare their response to nerve growth factor (7S NGF). SCG neurons required added NGF for their survival and a concentration of 1 μg/ml was found to be optimal. NG neurons, on the other hand, survived well for a long term without addition of NGF, but its application (1 μg/ml) was found to be effective in accelerating the growth of fibers (neurites) and neuronal somata. It is concluded that unlike SCG, NG neurons do not depend on exogenous NGF but may require an intrinsic trophic-like factor which may be contained in the serum of the medium, emanating from glial cells or by metabolic cooperation between neurons.     

Effects of nerve growth factor on the survival and synaptic function of Ia sensory neurons axotomized in neonatal rats

Sensory neurons with small diameters (A delta and C cells) are known to be responsive to exogenous NGF even at postnatal stages. We have examined whether large Group Ia sensory neurons (A alpha cells) arising from muscle spindles are also responsive to NGF in neonatal rats. For this purpose, monosynaptic excitatory postsynaptic potentials (EPSPs) were evoked in spinal motoneurons by Group Ia muscle afferent volleys. When a muscle nerve was crushed on the day after birth, the monosynaptic EPSPs elicited by afferent volleys from the muscle were depressed within several weeks. This synaptic depression was partially reversed by daily treatment with NGF. NGF treatment also enhanced the EPSPs evoked by stimulation of intact muscle nerves, but this effect was less marked than that on the EPSPs produced by stimulation of the previously crushed muscle nerve. Exogenous NGF was effective for the EPSPs when the treatment began on the day after birth but not when the treatment began 4 d after birth. Following crush of a muscle nerve on the day after birth, about 45% of the sensory neurons derived from the muscle were lost. The cell death of small sensory neurons was prevented by daily treatment with NGF, whereas the NGF treatment was ineffective in preventing the cell death of large sensory neurons. The results indicate that Group Ia sensory neurons are responsive to NGF during early postnatal life.     

Expression of nerve growth factor during the development of nervous system in early chick embryo

In the chick gastrula, nerve growth factor (NGF) is localized to the endoblast mesoblast presumptive head ectoderm but not in the presumptive neuroectoblast. During early morphogenesis the dorsal body ectoderm presumptive neural crest cells exhibit strong NGF positive cell surface reaction. NGF appears to be a marker of cells participating in morphogenetic movements but not early neural differentiation. NGF is localized where neural folds fuse and cells die allowing detachment of the neural tube from head ectoderm as well as in dead cells in the neurocoele. NGF reactivity in cells lining the evaginated extremities of the optic vesicle the floor of the neural tube the splanchnopleure heart primordia the inner outer surfaces of somites is suggestive of the role of NGF in primitive organ shaping.     

Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons

To analyze the possible influence of nerve growth factor (NGF) on neurofilament synthesis in primary sensory neurons, adjacent cryostat sections of lumbar dorsal root ganglia (DRG) from adult rats were processed for either NGF-receptor radioautography or in situ hybridization with a neurofilament cDNA probe. Labeling by both procedures was quantified with computer assistance for approximately 300 neurons in each of selected ganglia. For uninjured neurons, no correction was detected between NGF binding and neurofilament mRNA, even after infusion of NGF into the lumbar subarachnoid space for 1 week. One or 3 weeks after sciatic nerve transection, neurofilament labeling densities in large DRG neurons were sharply reduced and the normal bimodal pattern in frequency histograms had become unimodal. Intrathecal infusion of NGF counteracted this injury-induced reduction of neurofilament mRNA but only in neurons with high-affinity NGF receptors. To explain the effects of NGF on axotomized neurons and the normal diversity of neurofilament gene expression among neurons with NGF receptors, we postulate that NGF permits NGF-sensitive DRG neurons to respond differentially to a second factor stimulating neurofilament synthesis.     

Correlation between GAP43 and nerve growth factor receptors in rat sensory neurons

In mature rat sensory neurons, expression of the gene for the growth-associated protein, GAP43, was studied by in situ hybridization with a cDNA probe. Among neurons in normal lumbar dorsal root ganglia, labeling for GAP43 mRNA was heterogeneous, approximately one-half of the neurons being densely labeled. To characterize the latter population, individual neurons were examined in adjacent sections processed either for GAP43 hybridization or NGF-receptor radioautography. Virtually all neurons with high-affinity NGF binding sites had high basal levels of GAP43 mRNA and most GAP43-positive neurons bore NGF receptors. Another NGF-responsive population, sympathetic neurons in the superior cervical ganglion, also had high basal concentrations of GAP43 mRNA. Further co-localization studies in dorsal root ganglia were performed with immunohistochemistry for somatostatin and enzyme histochemistry for acid phosphatase. The latter 2 groups of sensory neurons have been previously shown to lack high-affinity receptors and were here shown to have low basal concentrations of GAP43 mRNA. From this and earlier studies, it can be assumed that substance P-immunoreactive neurons and strongly positive CGRP neurons synthesize GAP43 at high basal rate. One week following peripheral nerve transection, almost all neurons had high concentrations of GAP43 mRNA without correlation with NGF binding. Intrathecal infusion of NGF after the sciatic nerve was cut did not strongly influence this post-traumatic elevation in GAP mRNA. In normal dorsal root ganglia, neurons that have high-affinity NGF binding sites and are therefore potentially responsive to NGF also have high basal rates of synthesis of GAP43.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks, 141 +/- 36 pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20-30%, with 800-1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10-20% of neurons, mostly of small diameter (18-22 micron). SP content was low for 5 days then rose progressively after 14 days to 80-150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF 1185 +/- 176 pg/well vs NGF withdrawn day 8-12, 592 +/- 118 pg/well, mean +/- S.D., P less than 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.     

Expression and regulation of tyrosine hydroxylase in adult sensory neurons in culture: Effects of elevated potassium and nerve growth factor

To determine whether similar molecular mechanisms regulate the same proteins in diverse neuronal populations, the present study compared regulation of tyrosine hydroxylase (TOH) in placodal sensory and neural crest-derived sympathetic neurons in tissue culture. Long-term explant cultures of adult nodose and petrosal sensory ganglia (NPG) contained abundant TOH-immunoreactive neurons and exhibited TOH catalytic activity, as in vivo. After an initial decline during the first week of culture, enzyme activity was maintained at a stable plateau of 60% of zero time values for at least 3 weeks. However, exposure of 2-week-old cultures to depolarizing concentrations of potassium (K+; 40 mM) increased TOH activity approximately two-fold; total protein was unchanged, suggesting that the rise was due to increased TOH specific activity. Therefore, membrane depolarization in vitro appears to regulate this specific catecholaminergic (CA) trait in sensory, as in sympathetic CA cells. In sympathetic neurons, NGF regulates TOH activity throughout life. In marked contrast, TOH activity in adult NPG cultures was unchanged in the presence of 0, 10 or 100 units NGF/ml or in the presence of high concentrations of antiserum against the beta-subunit of NGF. Adult sympathetic neurons, however, grown under identical conditions, exhibited a 5- to 10-fold rise in TOH activity in the presence of NGF. Thus, unlike sympathetics, CA metabolism in adult NPG neurons is not regulated by NGF in vitro; NGF is therefore unlikely to mediate target effects on CA metabolism in placodal sensory neurons in vivo. Our findings indicate that certain mechanisms of CA regulation are shared by placodal sensory and neural crest-derived sympathetic neurons, whereas others are not.(ABSTRACT TRUNCATED AT 250 WORDS)     

人NGF促进感觉神经生长和诱导SP释放实验研究

目的采用体外模型对比研究观察,评估人痛性椎间盘细胞外基质对小鼠背根神经节神经轴突生长所产生的促进作用和神经肽（sP）的诱导作用。方法通过获取椎间盘源性下腰痛患者的椎间盘组织（退变组）,以及正常人腰椎间盘组织（正常组）提取细胞基质,对小鼠背根神经节神经元（DRG）进行培养,通过形态学观察神经元轴突生长和酶联免疫吸附法（ELISA）检测SP含量。结果实验组神经生长因子（NGF）浓度显著高于正常组;实验组神经元轴突平均长度为（134．17±2．21）μm,明显高于正常组的（121．28±4．10）μm,二者相比有明显差异性（P〈0．05）。在退变人椎间盘组织培养液中SP物质比率明显高于正常组（P〈0．001）,二者具有显著性差异。结论人退变椎间盘细胞外基质中高表达的NGF能促进感觉神经轴突的生长以及诱导和疼痛相关神经肽的释放。     

The response of chick sensory neurons to brain-derived neurotrophic factor

To determine the spectrum of activity of brain-derived neurotrophic factor (BDNF) among first-order sensory neurons, explants of the nine distinct populations of sensory neurons from embryonic chicks of 3-14 d incubation (E3-E14) were grown in collagen gels with and without BDNF in the culture medium. The explants responded to BDNF with profuse neurite outgrowth and comprised those in which neurons are derived from neural crest (the dorsomedial part of the trigeminal ganglion, rostromedial part of the trigeminal mesencephalic nucleus, jugular ganglion, and lumbosacral dorsal root ganglia) and from epibranchial placodes (the ventrolateral part of the trigeminal ganglion and the geniculate, petrosal, and nodose ganglia). This response was first clearly observed in the ventrolateral trigeminal and nodose ganglia as early as E4, but did not appear until later in other explants. The dorsomedial trigeminal, jugular, and geniculate ganglia were the latest to develop a response, which was not apparent until E8. In all explants the response was maximal between E10 and E12, and there was a decline in the magnitude of the response from E12 to E14. Although explants of the vestibular ganglion failed to respond to BDNF, the survival and growth of vestibular neurons in dissociated neuron-enriched cultures were promoted by BDNF. To investigate whether all first-order sensory neurons respond to BDNF or whether BDNF-responsive neurons comprise a distinct subset, we studied the influence of BDNF and NGF on the survival and growth of the placode-derived and the neural crest-derived neurons of the trigeminal ganglion in dissociated neuron-enriched culture.(ABSTRACT TRUNCATED AT 250 WORDS)