Commercial recombinant human beta-nerve growth factor and adult rat dorsal root ganglia contain an identical molecular species of nerve growth factor prohormone

Examination of commercial recombinant human beta-nerve growth factor (rh-beta-NGF) preparations with polyclonal antibodies specific to 13-kDa NGF and pro-NGF-specific domains revealed the presence of high-molecular-mass immunoreactive proteins, including a 60-kDa NGF prohormone. On incubation with a mixture of N- and O-specific glycosidases, the 60-kDa NGF pro-hormone generated a 32-kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF- and pro-NGF domain-specific antibodies also revealed the presence of high-molecular-mass proteins, including the 60-kDa NGF prohormone. Based on the presence of the 60-kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.     

Localization of basic fibroblast growth factor in a subpopulation of rat sensory neurons

Summary 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.     

In vitro effects of histamine liberator compound 48/80 on rat superior cervical ganglia with special regard to the small granule-containing cells

Summary The effect of the histamine liberator compound 48/80 on the rat superior cervical ganglia in vitro has been investigated. After incubation of the ganglia with compound 48/80: (1) ganglionic mast cells degranulate in the same manner as in other tissues; (2) cell bodies of the postganglionic neurons are not affected by compound 48/80; (3) there is evidence that ganglionic interneurons, the monoamine-containing cells are activated. The ultrastructural aspects of this process are characterized by degranulation of perikarya and accumulation of dense core vesicles in cell processes and in terminals adjacent to presynaptic membranes. These vesicles vary in size between 200–800 Å in diameter. They may represent storage sites of the neurotransmitter complexes that have undergone exocytosis. The results are discussed with special reference to models of exocytotic processes involving the adrenergic transmitter. It is concluded that monoamine-containing cells represent interneurons in sympathetic ganglia which inhibit ganglionic transmission and which are stimulated by low concentrations of compound 48/80 in vitro.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70 Hirnforschung).The authors wish to thank Professor Dr. J. Staubesand for his encouragement in the course of this work.Dedicated to Prof. Gian Töndury, Zurich, on the occasion of his 70th birthday.     

The granule-containing cell somata in the superior cervical ganglion of the rat,as studied by a serial sampling method for electron microscopy

Summary The surface of 4 granule-containing cells, in a cluster within the rat superior cervical ganglion, was studied by a serial sampling technique for electron microscopy. The result shows that all the 4 cells receive one, or three afferent synaptic boutons from the preganglionic fibers impinging upon their somata, and a somatic efferent synapse exists at two locations on each soma of the 2 of these cells. The postsynaptic element of the efferent synapse is observed to be represented by non-vesiculated and vesiculated segments of dendrites, soma and a possible axon collateral of the adrenergic principal neuron of the ganglion. There is a remarkably constant development of the attachment plaque between the granule-containing cells themselves, representing 1.7–2.3% of surface area for each cell. The surface area exposed to the extracellular space (covered only by a basal lamina) varies from 0.1 to 2.3% of the total perikaryal surface of the 4 cells. A tendency is noted that those cells without efferent synapses possess a more extensive area exposed to extracellular space than those forming somatic efferent synapse to the postganglionic elements.It is a pleasure to acknowledge the advice and encouragement of Professor A. Yamauchi throughout this work. I thank Mr. K. Kumagai and Miss K. Tsushida for their technical assistance.     

Induction of tyrosine hydroxlase synthesis in rat superior cervical ganglia in vitro by nerve growth factor and dexamethasone.

The addition of dexamethasone and nerve growth factor to organ cultures of superior cervical ganglia from young rats induces the synthesis of tyrosine hydroxylase. The combination of nerve growth factor and dexamethasone $\text{in vitro}$ produces a differential rate of tyrosine hydroxylase synthesis which approaches that obtained by the $\text{in vivo}$ administration of nerve growth factor.     

Age-dependence of the nerve fibre growth-promoting effects of hippocampus and exogenous nerve growth factor on cultured rat septum and superior cervical ganglion

Explants of hippocampus from rats at various ages evoked an intense nerve fibre growth from cocultured superior cervical ganglion and septum explants taken from newborn rats. The addition of antiserum to nerve growth factor (NGF) into the culture medium inhibited the outgrowth of nerve fibres from superior cervical ganglia, while septum explants still extended nerve fibres in the same medium.Septum explants responded to added NGF, as well as to cocultured hippocampus, during the first postnatal week only, whereas ganglia extended nerve fibres in NGF-containing cultures throughout the postnatal period and even at the age of 6 months if superoptimal concentration of NGF was used.The present results suggest that hippocampus releases NGF and some other growth factor(s) in culture throughout the postnatal period from birth to adulthood. On the other hand, the capacity of septum to extend nerve fibres in response to the growth factors appears to be restricted to the first postnatal week.     

The effects of nerve growth factor and anti-nerve growth factor antibody on the neuroendocrine reproductive system in the European starling Sturnus vulgaris

Thyroidectomy of starlings causes them to remain in breeding condition indefinitely; deactivation of gonadotrophin-releasing hormone neurons that is characteristic of photorefractoriness does not occur. We hypothesise that a neurotrophin, whose presence or ability to function is dependent upon thyroid hormones, is somehow involved in this termination of gonadotrophin-releasing hormone release. Nerve growth factor is one such candidate. Mouse 7S-nerve growth factor dissolved in artificial cerebro-spinal fluid was therefore infused into the lateral ventricle of thyroidectomised male starlings held on long days four times daily for 21 days and 31 days, in separate experiments, to see if photorefractoriness would occur. The result was significant gonadal regression in the treatment groups during the infusion period, with no change in testicular volume in the control groups. Testicular recrudescence occurred after the end of the treatment period. To see if this was a non-specific effect, or progression towards photorefractoriness per se, we used castrated, photorefractory starlings held on long days. Anti-nerve growth factor antibody was infused into the lateral ventricle at increasing concentrations and frequency. There was a significant rise in circulating luteinising hormone levels in the treatment groups as compared to controls, increasing with antibody dosage. Accepted: 30 January 1997     

The history of the discovery of the nerve growth factor

The Nerve Growth Factor (NGF) is the progenitor of a family of growth factors which is still expanding. The history of its discovery is very colorful; it is a rare combination of scientific reasoning, intuition, fortuities, and good luck. In addition, I believe that the collaboration of three scientists with very different backgrounds contributed to the success: I had grown up in a laboratory of experimental embryology, Dr. Levi-Montalcini came from neurology, and Dr. Stanley Cohen was from biochemistry. The decision where to begin the history of a discovery is always arbitrary. I shall give my reasons why I begin this story with my wing bud extirpations on chick embryos and the analysis of the effects of the operation on the development of spinal nerve centers, published in 1934. Of course, I am aware of the fact that the analysis of neurogenesis had been pioneered by Dr. R. G. Harrison and his students at Yale University since the beginning of this century. It should be mentioned that their experiments had been done on amphibian embryos. My own interest in problems of neurogenesis dates back to my Ph.D. thesis in the Zoology Department of Professor H. Spemann at the University of Freiburg in (the Federal Republic of) Germany; it dealt with the influence of the nervous system on the development of limbs in frog embryos. After I had obtained some inconclusive results I did the crucial experiment of producing nerveless legs. I removed the lumbar part of the spinal cord and the spinal ganglia before the outgrowth of nerve fibers. The nerveless legs developed normally in every respect, but the muscles atrophied eventually.     

蛇毒神经生长因子促进周围神经再生的诱发电位观察

目的：研究蛇毒神经生长因子（sNGF)对大鼠坐骨神经损伤后诱发电位的影响,评价蛇毒神经生长因子在促进周围神经再生中的作用。方法：建立大鼠坐骨神经钳夹模型,局部滴加药物和术后肌注sNGF,通过脊髓诱发电位（SEP）,运动诱发电位（MEP）评定,观察坐骨神经修复情况。结果：sNGF治疗后可使伤后SEP,MEP提早出现,结论：蛇毒提取的NGF对大鼠坐骨神经损伤修复具有促进作用。     

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.     

Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy

Summary Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules.No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.We wish to thank Dr. C. Rufener, Geneva, for communicating to us a new method of antibodyperoxidase coupling and Mrs. M. Durand-Wenger for her excellent technical assistance. This study was supported by the Swiss National Foundation for Scientific Research (Grant Nr. 3.432.74)     

Appearance of retrogradely labeled neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase conjugate into the contralateral ganglion

Summary Injection of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the superior cervical ganglion (SCG) of the rat results in accumulation of WGA-HRP in sympathetic postganglionic neurons in the contralateral SCG. The sympathetic pathways involved and the mechanism underlying the labeling were investigated. The labeling in neurons in the contralateral SCG was apparent 6 h after injection and increased in intensity with longer survival times. The number of labeled neurons reached 1300 at 72 h after the injection. Transection of the external (ECN) or internal carotid nerves (ICN) resulted in considerable reduction in the number of labeled neurons. Combined transection of both ECN and ICN virtually eliminated labeling in the contralateral SCG. This provides strong evidence that these two nerves are the major pathways for WGA-HRP transport out of the SCG. No labeling was observed in the contralateral SCG following injection of horseradish peroxidase (HRP). Therefore, it seems unlikely that a direct nerve connection exists between the bilateral ganglia. Instead, the labeling of contralateral SCG neurons appears to depend on the transneuronal transport capacity of WGA-HRP, which conveys the marker in an anterograde direction along the postganglionic fibers to terminals in sympathetic target organs, and then delivers it transneuronally to contralateral SCG neurons. We suggest that the sympathetic nerve fibers originating in the bilateral SCGs run intermingled and are in close contact in their peripheral target organs.     

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

The effects of nerve growth factor on polyamine metabolism in PC12 cells

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.     

腺病毒介导人肝细胞生长因子对大鼠皮层神经元损伤的保护作用

目的:探讨腺病毒介导人肝细胞生长因子(Ad-HGF)对体外无血清培养所致大鼠皮层神经元损伤的保护作用.方法:用流式细胞仪测定不同感染强度(MOI)条件下携带绿色荧光蛋白的重组腺病毒(Ad-GFP)(25,50,100,200pfu/cell)对皮层神经元的转染效率,确定最佳MOI;ELISA测定Ad-HGF在最佳感染强度下皮层神经元中的表达规律;通过中性红染色和PI-Hoechst33342双染色法比较转染Ad-HGF组与对照组(转染Ad-GFP组和空白对照组)间无血清培养后6 h、12 h、24 h和48 h细胞的存活情况.结果:在感染强度为50 pfu/cell时,重组腺病毒对皮层神经元的转染率达99.3%,Ad-HGF能在皮层神经元中有效而持久地表达,接种后2 h转染Ad-HGF组的细胞死亡率和凋亡率在无血清培养后12 h明显低于两对照组(P＜0.05).结论:Ad-HGF对接种后2 h转染组无血清培养所致的损伤有显著的保护作用,对接种后第5 d转染的皮层神经元无血清所致的损伤虽亦具有一定的保护作用,但不明显.     

Forelimb muscle activity following nerve graft repair of ventral roots in the rat cervical spinal cord

Current research on the cellular mechanisms of nerve regeneration suggests the application of nerve growth factors at the repair sites to be beneficial. To test the effectiveness of this approach, we performed transections of the C6 and C7 ventral rootlets from their original sites in the spinal cord of 18 rats. We investigated the electrophysiological changes in three groups of rats operated on by different repair strategies. Six rats comprised the control group (G1). In the other 12 rats, 24 rootlets were implanted into the spinal cord by means of an intercostal nerve graft through the pia mater immediately after transection. Six rats (G2) had fibrin glue applied at the incision. The last 6 rats (G3) had grafts with acidic fibroblast growth factor (aFGF) added to the fibrin glue. The rats' functional recovery was evaluated electrophysiologically at 6 weeks and 6 months after the operation. Needle electromyography showed profound fibrillation potentials (Daube's scoring system) in the deltoid, biceps, and triceps of the operated forelimbs in all groups 6 weeks after the operation. After 6 months, there was a significant decrease in the amount of fibrillation potentials in all groups (G1, G2 and G3, p < 0.0001, 0.0001, 0.0009, respectively, generalized estimating equation, repeated measures) and a significantly high probability for motor units present in sampled muscles of G2 and G3 as compared to G1 (log odds ratio in G2 = 51.8316, G3 = 57.4262, generalized estimating equation). We conclude that several cervical roots can regenerate through intercostal nerve grafts applied using fibrin glue. Adding aFGF may increase the efficacy of sprouting.     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK_d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K_d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.