Nerve growth factor stimulates growth of cortical pyramidal neurons in young adult rats

The cytoarchitectonics of pyramidal neurons in the cerebral cortex of non-lesioned rats can be re-modeled by i.c.v. infusions of nerve growth factor (NGF). 4 months after the application of NGF, the pyramidal neurons in layers III and V of the motor cortex and layer V of the anterior cingulate cortex were analyzed and compared with pyramidal neurons from vehicle-treated rats. NGF-treated brains showed: (1) significant increase in dendritic branching in the basilar fields of the layer V, but not layer III, neurons; and (2) a significant increase in spine density in the terminal, but not proximal, dendritic branches. These findings indicated that, besides its known effects on forebrain cholinergic neurons, NGF produces a very generalized synaptic re-modeling involving the cells responsible for the major output of the cerebral cortex in the intact adult brain. © 1979 Elsevier Science B.V. All rights reserved.     

Sequestration requirements for the degradation of 125I-labeled β nerve growth factor bound to embryonic sensory neurons

Nerve growth factor interacts with responsive cells by binding to cell surface membrane receptors. There are two different receptors on both embryonic sensory and sympathetic neurons, a high-affinity (type I) receptor and a lower-affinity (type II) receptor. Sequestration, which we have defined as bound nerve growth factor that becomes inaccessible to the external milieu with time, occurs through the type I receptor on both sensory and sympathetic neurons. We describe here a process subsequent to sequestration involving internalization and degradation of bound nerve growth factor and showing that bound nerve growth factor is not degraded under the following conditions: (1) low temperature, ie 4°C; (2) when a large excess of unlabeled nerve growth factor is added concomitantly with the labeled nerve growth factor and the temperature is raised from 4°C to 37°C; (3) when metabolic inhibitors sodium fluoride and dinitrophenol are added concomitantly with the labeled nerve growth factor and the temperature is raised from 4° to 37°C. On the other hand, conditions that allow bound nerve growth factor to be degraded are the following: (1) incubation of the sensory nerve cells at low temperature (ie, 4°C) only in the presence of labeled nerve growth factor, then raising the temperature to 37°C; (2) when sodium fluoride and dinitrophenol are added when the temperature is raised to 37°C; (3) when excess unlabeled nerve growth factor is added when the temperature is raised to 37°C. These studies are consistent with the idea that nerve growth factor has to bind to the cells in order to be degraded; however, binding is not sufficient for degradation to occur. Second, the bound nerve growth factor must be sequestered in order to be degraded. Third, the process of internalization of the bound nerve growth factor, unlike sequestration, is not an energy-dependent process. Thus, it seems reasonable to suggest the following steps for the interaction of nerve growth factor with responsive cells: binding to a cell surface membrane receptor, followed by sequestration of the bound nerve growth factor, and finally, internalization of the sequestered nerve growth factor.     

Characterization of nerve growth factor binding to embryonic rat spinal cord neurons

The binding of iodinated beta-nerve growth factor, [125I]-NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 x 10(-11) M and 4 x 10(-9) M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]-NGF binding to each metrizamide fraction showed that the motoneuron-enriched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.     

Decrease in the number of lower affinity (type II) nerve growth factor receptors on embryonic sensory neurons does not affect fiber outgrowth

Nerve growth factor binds to two different specific receptors on responsive cells. The relationship of these two receptors is not fully understood at this time. We have studied the binding of labeled NGF to a different strain of white leghorn chicken embryo dorsal root ganglionic cells. The equilibrium dissociation constants for the two sites (K = 4.1 ± 1.8 × 10^?11M, K = 1.0 ± 0.8 × 10^?9M) are identical to those obtained previously. Also, the number of type I sites per cell (3.8 ± 1.3 × 10³) is the same as that previously determined. However, the number of type II sites per cell (1.9 ± 1.3 × 10⁴) is significantly different than that previously determined. This 2.5-fold decrease in the number of type II sites does not affect the concentration of NGF needed to obtain maximal fiber outgrowth from explanted sensory ganglia. The rate of association (1.2 ± 0.2 × 10⁷ M^?1 sec^?1 at 22°C) of labeled NGF with receptors on sensory neurons from this different strain of chickens is identical to that previously obtained. The rate of association of NGF with its receptors on sensory neurons was also determined at 4°C. This rate constant (2.1 ± 1.1 × 10⁶ M^?1 sec^?1) along with the rate constants obtained at 22° and 37°C were used to determine an activation energy for the binding of NGF to its receptors. The activation energy obtained (16.2 kcal/mole) suggests that binding is not a diffusion-controlled process.     

Time and dose dependencies of effects of nerve growth factor on sympathetic and sensory neurons in neonatal rats

Nerve growth factor (NGF) plays a role in the development of several components of the sympathetic and sensory nervous systems. The objectives of this study were to examine the time and dose dependencies of some of the well known effects of NGF on sympathetic ganglia and to examine qualitatively and quantitatively the recently described effects on sensory ganglia of neonatal rats. Single doses of NGF as low as 0.1 mg/kg produce increases in tyrosine hydroxylase (TOH) activity in superior cervical ganglia (SCG), and doses of 3 mg/kg produce maximal effects. Larger doses and longer treatments are required to see increases in protein content of the SCG. Larger doses are also required to affect TOH activity in the adrenal gland. Increases in TOH activity in SCG can be observed within 18 h of injection. Chronic NGF treatment for three weeks produces no change in blood pressure or heart rate in neonatal rats. Chronic administration of NGF (1 or 3 mg/kg/day) results in dose-related increases in the protein content of dorsal root ganglia (DRG). The increase in protein content of the DRG was associated with an increase in the diameter of smaller neurons (those<30 μm in diameter), but NGF caused no change in the number of neurons.     

Nerve growth factor and injured peripheral nerve regeneration

Nerve growth factor （NGF） exhibits many biological activities, such as supply of nutrients, neuroprotection, and the generation and rehabilitation of injured nerves. The neuroprotective and neurotrophic qualities of NGF are generally recognized. NGF may enhance axonal regeneration and myelination of peripheral nerves, as well as cooperatively promote functional recovery of injured nerves and limbs. The clinical efficacy of NGF and its therapeutic potentials are reviewed here. This paper also reviews the latest NGF research developments for repairing injured peripheral nerve, thereby providing scientific evidence for the appropriate clinical application of NGF.     

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.     

Epidermal growth factor influences the neurotrophic/differentiating action of nerve growth factor

Exposure of naive PC12 cells, sympathetic neurons from rat superior cervical ganglia, and brain-derived septal neurons to epidermal and nerve growth factors simultaneously resulted in some alteration of cellular events induced by nerve growth factor alone. A more pronounced decline of catecholamine content, no additional change in acetylcholinesterase activity, and additive stimulation of RNA and protein syntheses were found in PC12 cells. Earlier elevation of the enzyme activity was observed in sympathetic but not in septal neurons. Epidermal growth factor appeared to support independently the same level of acetylcholinesterase activity in septal neurons as revealed for nerve growth factor during the first week and cell survival throughout 2 weeks of observation. The data obtained indicate that epidermal growth factor augments temporarily some effects of nerve growth factor, thus supporting the idea of an important role of mitogenic growth factors in neural development as complementary and/or substitutive regulators of nerve cell differentiation and survival.     

Nerve growth factor induces Fos-like immunoreactivity within identified cholinergic neurons in the adult rat basal forebrain

Immunocytochemical techniques were used to examine and compare the effects of intracerebroventricular administration of nerve growth factor (NGF) on Fos expression within identified cholinergic and non-cholinergic neurons located in different regions of the adult rat basal forebrain. Animals were killed 1, 3, 6, and 12 h after receiving NGF (0.5 or 5.0 μg) or vehicle into the left lateral ventricle and sections through the medial septum, diagonal band of Broca, nucleus basalis magnocellularis, and striatum were processed for the combined immunocytochemical detection of Fos and choline acetyltransferase (a marker for cholinergic neurons), or Fos and parvalbumin (a marker for gamma aminobutyric acid (GABA)-containing neurons). NGF produced a significant increase in the percentage of cholinergic neurons containing Fos-like immunoreactivity within all four regions examined. The largest increases were detected in the medial septum (47.8%) and the horizontal limb of the diagonal band of Broca (67.7%). In these areas, NGF-mediated induction of Fos-like immunoreactivity was detected as early as 3 h, peaked at 6 h, and was reduced by 12 h, postinfusion. Small but significant increases in the percentage of cholinergic neurons containing Fos-like immunoreactivity were also detected in the striatum (4.2%) and in the nucleus basalis magnocellularis (19.2%) 3–12 h following administration of the higher dose of NGF. No evidence for an NGF-mediated induction of Fos within parvalbumin-containing neurons was detected in any of the four regions at any of the time-points examined; however, evidence for an NGF-mediated induction of Fos within epithelial cells lining the lateral ventricle was observed. These data demonstrate that NGF induces Fos expression within cholinergic, and not parvalbumin-containing (GABAergic), neurons in the basal forebrain, and furthermore that intracerebroventricular administration of NGF influences the different subgroups of basal forebrain cholinergic neurons to different degrees. ©1977 Elsevier Science B.V. All rights reserved.     

Nerve growth factor receptor-immunoreactive neurons within the developing human cortex.

A monoclonal antibody recognizing the p75 receptor for nerve growth factor (NGF) was used to assess the immunohistochemical expression of NGF receptors within the developing human neo-, limbic, and paralimbic cortices as well as the hippocampal complex. Between embryonic weeks 16 and 26, a transient population of neurons located within the upper and lower subplate zones of the neo-, limbic, and paralimbic cortices expressed the receptor for NGF. In contrast, NGF receptor-immunoreactive neurons were only observed in the upper subplate zone of the entorhinal cortex at embryonic week 40 (term), a staining pattern not observed in a 5-year-old specimen. The expression of NGF receptor-immunoreactive neurons within the upper subplate zone between embryonic weeks 16 and 40 was characterized by a dense band of immunoreactive neurons and neuropil. These neurons were bipolar with basal and apically directed neurites. NGF receptor-immunoreactive neurons were also scattered throughout the lower subplate zone and underlying white matter between embryonic weeks 19 and 26. These neurons were multipolar, with less apically directed neurites. NGF receptor-immunoreactive subplate neurons displayed a topographic distribution with the heaviest concentration found within limbic and paralimbic cortices as well as association neocortex. In contrast, light to moderate NGF receptor-immunoreactivity was seen in sensory-motor cortex. Within the hippocampal complex, only a few lightly stained NGF receptor-immunoreactive neurons were seen within the fimbria, hilar region of the dentate gyrus, and subiculum. The expression of NGF receptor-immunoreactivity increased within the subplate zone of the pre- and parasubiculum culminating in intense entorhinal cortex staining. As the entorhinal cortex merged with the developing inferior temporal association cortex, there was a marked reduction in staining intensity. In contrast to those in the subplate zone, neurons within the germinal zone and cortical plate were NGF receptor immunonegative at all times examined. The presence of NGF receptors in the subplate zone suggests that neurotrophins such as NGF play an important role in the transient viability of these neurons as well as in the guidance of cortical afferent inputs into topographically organized regions of the cerebral cortex.     

Immunohistochemical phenotype of sensory neurons associated with sympathetic plexuses in the trigeminal ganglia of adult nerve growth factor transgenic mice

Following peripheral nerve injury, postganglionic sympathetic axons sprout into the affected sensory ganglia and form perineuronal sympathetic plexuses with somata of sensory neurons. This sympathosensory coupling contributes to the onset and persistence of injury-induced chronic pain. We have documented the presence of similar sympathetic plexuses in the trigeminal ganglia of adult mice that ectopically overexpress nerve growth factor (NGF), in the absence of nerve injury. In this study, we sought to further define the phenotype(s) of these trigeminal sensory neurons having sympathetic plexuses in our transgenic mice. Using quantitative immunofluorescence staining analyses, we show that the invading sympathetic axons specifically target sensory somata immunopositive for several biomarkers: NGF high-affinity receptor tyrosine kinase A (trkA), calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NFH), and P2X purinoceptor 3 (P2X3). Based on these phenotypic characteristics, the majority of the sensory somata surrounded by sympathetic plexuses are likely to be NGF-responsive nociceptors (i.e., trkA expressing) that are peptidergic (i.e., CGRP expressing), myelinated (i.e., NFH expressing), and ATP sensitive (i.e., P2X3 expressing). Our data also show that very few sympathetic plexuses surround sensory somata expressing other nociceptive (pain) biomarkers, including substance P and acid-sensing ion channel 3. No sympathetic plexuses are associated with sensory somata that display isolectin B4 binding. Though the cellular mechanisms that trigger the formation of sympathetic plexus (with and without nerve injury) remain unknown, our new observations yield an unexpected specificity with which invading sympathetic axons appear to target a precise subtype of nociceptors. This selectivity likely contributes to pain development and maintenance associated with sympathosensory coupling.     

Nerve growth factor and neural oncology

The precise role of the nerve growth factor protein (NGF) during the growth and development of the human nervous system is not determined. Although it appears to influence a number of neural functions, its mechanism of action is poorly understood. A number of researchers have proposed that NGF may be involved in several pathological conditions including cancer. It has been shown that NGF is secreted by certain sarcoma (23), neuroblastoma (113), and glioma (7,102,136) cell lines and can bind to neuroblastoma and metastatic melanoma cell lines (42). Neuroblastoma (136,181) and pheochromocytoma (165) cells in vitro can be induced by NGF to differentiate toward a morphologically "more benign" state and appropriate NGF treatment of rats can reduce the number of chemically induced gliomas and neurinomas (174,178). NGF can also reduce the growth of intracerebrally inoculated anaplastic glioma cells (172). Anti-NGF treatment of rats (178) and mice (179) can alter the tumor distribution observed following ethylnitrosourea or benzo(a)pyrene treatment (10). In humans, it has been reported that serum levels of NGF are usually elevated in persons "at risk" for neurofibromatosis (156). The precise nature of the NGF role is not known in these instances. Further understanding of the action of NGF could be of clinical importance.     

Nerve conduits and growth factor delivery in peripheral nerve repair

Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Transected peripheral nerves can be bridged by direct surgical coaptation of the two nerve stumps or by interposing autografts or biological (veins) or synthetic nerve conduits (NC). NC are tubular structures that guide the regenerating axons to the distal nerve stump. Early synthetic NC have primarily been made of silicone because of the relative flexibility and biocompatibility of this material and because medical-grade silicone tubes were readily available in various dimensions. Nowadays, NC are preferably made of biodegradable materials such as collagen, aliphatic polyesters, or polyurethanes. Although NC assist in guiding regenerating nerves, satisfactory functional restoration of severed nerves may further require exogenous growth factors. Therefore, authors have proposed NC with integrated delivery systems for growth factors or growth factor-producing cells. This article reviews the most important designs of NC with integrated delivery systems for localized release of growth factors. The various systems discussed comprise NC with growth factors being released from various types of matrices, from transplanted cells (Schwann cells or mesenchymal stem cells), or through genetic modification of cells naturally present at the site of injured tissue. Acellular delivery systems for growth factors include the NC wall itself, biodegradable microspheres seeded onto the internal surface of the NC wall, or matrices that are filled into the lumen of the NC and immobilize the growth factors through physical-chemical interactions or specific ligand-receptor interactions. A very promising and elegant system appears to be longitudinally aligned fibers inserted in the lumen of a NC that deliver the growth factors and provide additional guidance for Schwann cells and axons. This review also attempts to appreciate the most promising approaches and emphasize the importance of growth factor delivery kinetics.     

Identification of two phosphoproteins affected by serotonin in Aplysia sensory neurons

Protein phosphorylation appears to play important roles in the mechanisms responsible for presynaptic facilitation in Aplysia. To screen for phosphoproteins that may be involved in facilitation, we previously examined protein phosphorylation in pleural sensory neurons as a function of different durations (2 min, 25 min and 1.5 h) of serotonin treatments. Different durations of serotonin had unique effects on the phosphorylation of different sets of proteins. To determine the functions of these phosphoproteins, we have begun to obtain their amino acid sequences using protein microsequencing techniques. We report here partial sequencing of 2 such proteins. One protein (S6), whose phosphorylation was affected by 2 min treatments with serotonin, appeared to be an intermediate filament protein. Another protein (L55), whose phosphorylation was affected by 1.5-h treatments with serotonin, appeared to be a calmodulin-like Ca²⁺-binding protein. Although the exact cellular functions for S6 and L55 are not known, obtaining partial sequences of these proteins sets the stage for future studies that will examine their regulation and their specific roles in facilitation.     

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.     

Na+, K+-ATPase and ouabain binding activities of chick embryo dorsal root ganglia supported by and deprived of nerve growth factor

Recently we have shown that nerve growth factor (NGF) influences the coupled movements of Na⁺ and K⁺ across the membrane of chick embryo dorsal root ganglion (DRG) and other target cells. These ionic effects of NGF are consistent with a model in which NGF acts through the classical Na⁺,K⁺-ATPase pump. Direct evidence for NGF-induced alteration of this pump has been sought in the present study through two approaches. In one approach, DRG cell dissociates were incubated for 6 hours without NGF (to allow development of the ionic defect), and [³H]ouabain binding to the cells was measured before and during a delayed administration of NGF. No differences were detected in either total binding or binding time. In the second approach, intact DRG or DRG dissociates were incubated for 6 hours with or without NGF, or received NGF after 6 hours of deprivation, and Na⁺,K⁺-ATPase (ouabain-sensitive) activity was measured in the corresponding microsomal preparations. Activity levels were found to be the same in all cases, and were unchanged by addition of NGF directly to the enzyme preparations. Different concentrations of Na⁺, K⁺, or ATP affected in identical manners the enzyme preparations from NGF-treated and NGF-deprived ganglia, speaking against NGF-imposed changes in the affinities of the corresponding enzyme sites. Also unsuccessful were attempts to reveal NGF-related differences by testing ouabain-sensitive ATPase activity (1) in the presence of varying concentrations of cyclic AMP or of Ca²⁺, (2) after treatment with Triton X--100 or in the presence of vanadate, or (3) on addition of a 100,000g DRG extract. These negative findings are discussed in terms of the Na⁺,K⁺-ATPase hypothesis for NGF action.     

大鼠坐骨神经修复后重组睫状神经营养因子对相关神经元生长相关蛋白43、生长抑素表达的影响

目的观察周围神经修复后，重组睫状神经营养因子(CNTF)对相关神经元中生长相关蛋白表达的调控作用。方法用硅管套接切断的成年大鼠坐骨神经，在神经切断处给予重组CNTF，用免疫组织化学和原位杂交组织化学方法结合计算机图像分析观测L4脊髓和L4、L5脊神经节中生长相关蛋白43(GAP43)和生长抑素(SOM)mRNA的相对含量。结果在CNTF组修复侧脊髓前角外侧核，大、中型神经元胞质中神经元GAP43阳性物质的面积百分率显著高于生理盐水组，SOM mRNA杂交信号阳性的大、中型神经元的数量少于生理盐水组，但两组脊神经节的相应指标无显著差别。结论坐骨神经修复后，外加重组CNTF能上调相关运动神经元表达GAP43，下调其表达SOM mRNA，但对感觉神经元的相应作用不明显。