Nerve fiber production by intraocular adrenal medullary grafts: Stimulation by nerve growth factor or sympathetic denervation of the host iris

Summary This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.     

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.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues.

We have developed a sensitive assay for the quantification of nerve growth factor mRNA (mRNANGF) in various tissues of the mouse using in vitro transcribed RNANGF. Probes of both polarities were used to determine the specificity of the hybridization signals obtained. Comparison of NGF levels with its mRNA revealed that both were correlated with the density of sympathetic innervation. Thus, vas deferens contained high levels of both NGF and mRNANGF, whereas skeletal muscle levels were barely detectable, indicating that in peripheral tissues NGF levels are primarily regulated by the quantity of mRNANGF and not by the rate of processing of NGF precursor to NGF. However, although superior cervical ganglia contained the highest levels of NGF, its mRNA was barely detectable. Thus, the high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. The quantity of NGF found in the submandibular glands of female animals was three orders of magnitude higher than expected from their mRNA levels. This observation is discussed in the context of the difference between the mechanism of storage and exocytosis of exocrine glands versus the constitutive release from other tissues.     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.     

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.     

Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.     

Effect of ten different target tissues on nerve fibre outgrowth from rat sympathetic ganglia in organotypic co-cultures

Sympathetic thoracic chain ganglia of 3-day-old rats were cultured in collagen gel medium for 24 hours together with explants from heart atrium, liver, kidney, cornea, iris, lung, adrenal cortex, adrenal medulla, skeletal muscle, or vas deferens. The extent of nerve fibre growth was estimated by counting the number of fibres crossing each arc of a sector drawn in the ocular. The various tissues stimulated nerve fibre growth to distinctly different extents. The increase in the nerve fibre outgrowth induced by atrium and iris was statistically highly significant. Kidney, liver, vas deferens, lung, and adrenal cortex had, in that order, a decreasingly stimulatory influence on sympathetic chain ganglia. Yet they all caused a significant increase in nerve fibre growth. Skeletal muscle, cornea and adrenal medulla had no stimulatory effect. Since the significant effects of the tissue explants were abolished by antiserum to nerve growth factor (NGF), it is concluded that the observed effects were due to NGF produced by the explants. The only exception was vas deferens, the stimulatory action of which proved to be partially NGF-independent.     

神经生长因子在6－OHDA化学性去交感神经中的保护作用

本实验用６－ＯＨＤＡ造成成年小白鼠领下腺化学性去交感神经,观察了神经生长因子对该神经的保护作用。６－ＯＨＤＡ（１５ｍｇ／ｋｇｉｐ）处理后２４ｈ腺体内去甲肾上腺素（ＮＥ）含量降至正常水平的２％以下。若在６－ＯＨＤＡ处理同时开始多次给予神经生长因子（ＮＧＦ）,则ＮＥ残留量明显提高。减小６－ＯＨＤＡ剂量至１０ｍｇ／ｋｇ,ＮＥ残留量增加,同时ＮＧＦ的作用亦较用６－ＯＨＤＡ１５ｍｇ／ｋｇ时更为显著。若提前２４ｈ给予ＮＧＦ,尽管仍显著提高ＮＥ残留量,但程度却显然低于与６－ＯＨＤＡ同时给予者。以上结果表明外源性ＮＧＦ对６－ＯＨＤＡ造成交感神经化学性损毁有保护作用,此作用与神经受损的严重程度以及ＮＧＦ处理时间有关。     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Nerve growth factor-induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF-induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP-43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF-induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75NTR expression. We propose that p75NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF.     

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.     

Nerve growth factor synthesis in cultured rat iris: modulation by endogenous transmitter substances

Organ cultures of rat iris show a characteristic change in the levels of both nerve growth factor (NGF) and its mRNA: a rapid but transient initial increase is followed by a smaller but persistently elevated NGF synthesis. This time course may be influenced by release of a factor(s) from degenerating nerve terminals and/or by the lack of some factor(s) repressing NGF synthesis in vivo. We therefore analyzed the influence of biogenic amine transmitter substances and putative neuropeptides on this elevation of NGF synthesis in cultured iris. The marked increase of NGF synthesis seen initially in culture was not completely mimicked by any of the substances tested. A specific increase in NGF production up to 150% of control was observed only with cGMP. We also obtained some evidence that reaction to trauma following the culture procedure could enhance NGF production: cutting of irides into small pieces increased NGF production in culture up to 250% of control and, vice versa, treatment with 1 microM dexamethasone decreased NGF production to about 60% of control. However, the sympathetic neurotransmitter norepinephrine (NE) decreased both NGF and its mRNA levels specifically in a dose-dependent manner (0.01-1 mM) to a minimum of about 25% of control. In situ hybridization with mRNA(NGF)-specific probes showed that in cultures of dissociated iris cells all cells were capable of expressing mRNA(NGF), but that 0.1 mM NE preferentially decreased expression of mRNA(NGF) in smooth muscle cells. Thus, our results indicate that the sympathetic transmitter NE is capable of downregulating NGF synthesis in the target cells of sympathetic neurons.     

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.     

Studies on the action of nerve growth factor: I. Characterization of a simplified in vitro culture system for dorsal root and sympathetic ganglia

Neurite outgrowth from dorsal root (DRG) and sympathetic ganglia has been studied utilizing a simplified in vitro culture system for intact ganglia. Attachment of ganglia to tissue culture plates was achieved after a brief incubation of ganglia on the plates in the presence of 100% fetal calf serum or 5% ovalbumin in F12 medium. Neurite outgrowth from dorsal root and sympathetic ganglia was dependent on the continued presence of nerve growth factor (NGF) and on the NGF concentration. The NGF induced neurite outgrowth from DRG cultured in serum-free medium was delayed approximately 24 hr compared to the outgrowth in serum-containing medium.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Nerve growth factor–induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF‐induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75^NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75^NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP‐43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF‐induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75^NTR expression. We propose that p75^NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 51–66, 1999     

Properties of the nerve growth factor receptor of a clonal line of rat pheochromocytoma (PC12) cells.

The interaction of nerve growth factor (NGF) with its receptor on cells of the PC 12 cell line was studied. All experiments were done at 0.5 °C to minimize degradation and processes requiring membrane mobility. Under these conditions, a single class of high affinity binding sites with a dissociation constant of 2.9 × 10^?9 M was observed. The number of receptors per cell was 58000. The binding was linear with the number of cells in the assay and was not displaced by proteins other than native nerve growth factor. Trypsin treatment of the cells destroyed the specific binding. The removal of divalent cations had no effect on the binding. Culturing the cells for 2 weeks in NGF prior to assay did not change the receptor number or receptor affinity and there was a similar lack of effect of the density of the culture from which the cells were taken for assay. The present findings are compared with previous studies on the dorsal root ganglia and sympathetic ganglia neurons, and the implication for the use of PC 12 as a model for the study of NGF action are discussed.     

Nerve growth factor-mediated induction of tyrosine hydroxylase in rat superior cervical ganglia in vitro.

Exposure of rat sympathetic ganglia to 3 microgram/ml of 2.5 S nerve growth factor (NGF) resulted in a 100% increase in tyrosine hydroxylase activity within 48 h. Pulselabeling of proteins with [3H]leucine, followed by immunoprecipitation with antibodies to tyrosine hydorxylase and isolation of the precipitated enzyme by gel electrophoresis, demonstrated that the increase in tyrosine hydroxylase activity was due to enhanced de novo synthesis. The incorporation of [3H]leucine into tyrosine hydroxylase was increased by 150% compared to a 17% increase in total protein synthesis, which was not statistically significant. The fact that the half-life of pulse-labeled tyrosine hydroxylase was the same for NGF-treated and control organ cultures of superior cervical ganglia excludes the possibility that enhanced tyrosine hydroxylase labeling by NGF is due to decreased degradation. We conclude that, without modulatory factors which play a role in vivo, NGF can enhance the synthesis of tyrosine hydroxylase in sympathetic ganglia in vitro, provided organ culture conditions which permit optimal survival of adrenergic neurons are selected.