Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

beta-Nerve growth factor (beta NGF) receptors on glial cells. Cell-cell interaction between neurones and Schwann cells in cultures of chick sensory ganglia.

Receptors for beta-nerve growth factor (beta NGF), so far regarded as specific cell surface markers of certain peripheral neurones, were found to be expressed on cultured non-neuronal cells of chick embryo dorsal root ganglia (drg) (Kd beta NGF = 2 X 10(-9) M). Autoradiography revealed that binding of [125I] beta NGF was restricted to a subpopulation of the non-neuronal drg cells. Cultured embryonic skin fibroblasts, liver cells, gut cells, muscle fibroblasts, myoblasts, and myotubes, as well as macrophages and the cell lines 3T3, 3T3SV40, BHK, BHK Py, PCC3 and ND1, did not express receptors for beta NGF. Non-neuronal drg cells obtained by a procedure designed for the preparation of pure Schwann cells, as well as RN6 Schwannoma cells, were beta NGF receptor positive. The beta NGF receptor-positive non-neuronal drg cells displayed behaviour typical of Schwann cells in their interaction with drg neurones in single cell, as well as explant cultures. Three stages of neurone-Schwann cell interaction were discernible: (1) association--neurites preferentially grew over beta NGF receptor-positive non-neuronal cells; (2) cell division/alignment--beta NGF receptor-positive non-neuronal cells were induced to proliferate and aligned and elongated along neurites; (3) ensheathment--the outline of beta NGF receptor-positive non-neuronal cells and neurites merged. In drg cell cultures prepared from embryonic stages E6-E10, 25-40% of the non-neuronal cells were beta NGF receptor-positive. Later in development, from E12 onward, less than or equal to 1% of the cultured non-neuronal cells expressed beta NGF receptors.     

Nerve Growth Factor Stimulates Phospholipid Methylation in Target Ganglionic Neurons Independently of the Cyclic AMP and Sodium Pump Responses

Suspensions of neurons prepared from embryonic day 12 (E12) chick sympathetic ganglia were incubated with [methyl-3H]methionine in the absence of nerve growth factor (NGF). Presentation of the factor for different periods of time resulted in an approximate three-fold stimulation of radioactivity incorporated into total phospholipid, followed by a rapid decline thereafter. Both the magnitude and the time of the response were dependent on the NGF concentration used. Also examined were possible relationships of phospholipid methylation to two other short-latency responses to NGF, i.e., control of the Na+,K+-pump and elevation of cyclic AMP content. Incubation of E12 sympathetic neurons with known transmethylase inhibitors (shown to be active in the present system) failed to prevent reactivation of the Na+,K+-pump in response to NGF administration. E16 sympathetic neurons and E15 sensory neurons, which do not depend on exogenous NGF for control of their Na+,K+-pump, still show a stimulation of phospholipid methylation when challenged with the factor. Blockage of the pump with ouabain also fails to prevent a methylation response. Thus, the pump and methylation responses to NGF occur independently of each other. Intact E8 chick dorsal root ganglia, but not E12 sympathetic ganglia, display a rapid and transient rise in their cyclic AMP content when presented with NGF. At a concentration of 10 biological units/ml, NGF elicits a peak of phospholipid methylation at 4 min, and a peak of cyclic AMP at 10 min. Methylation inhibitors prevent the methylation response, but not that of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

Loss of nerve growth factor receptors in sympathetic ganglia from aged mice

[125I]-Nerve growth factor (NGF) binding was studied in superior cervical ganglia from mice 4-7 months and 24-27 months of age. Scatchard analysis demonstrated losses of both high and low affinity components of NGF receptor. These results indicate loss of NGF receptors may lead to the diminished responsiveness to NGF in aged sympathetic ganglion neurons.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Specific binding of covalently cross-linked mouse nerve growth factor to responsive peripheral neurons.

The binding characteristics of [¹²⁵I]nerve growth factor, covalently cross-linked with dimethyl suberimidate, to chick embryonic dorsal root ganglia are indistinguishable from the iodinated native hormone. Both show non-saturability, non-linear Scatchard plots and acceleration of dissociation of hormone-receptor complexes by native hormone which is reflected in the binding constants calculated. These results demonstrate that dimerization of the native hormone at the receptor is not responsible for the negatively cooperative behavior observed for native nerve growth factor. Further, experiments with amino-silylated glass tubes also eliminate interaction between hormone and reaction vessel as an explanation of the non-saturable and multiple affinity properties of the observed binding.     

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.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Development of trophic interactions in the vertebrate peripheral nervous system

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of thetrk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.     

The effect of nerve growth factor (NGF) on the synthesis of gangliosides

—Dorsal root ganglia from 8-day- and 14-day-old chick embryos contained gangliosides with a pattern qualitatively similar to that of embryonic chick brain. The pattern of gangliosides from dorsal root ganglia changed with age, there being a decrease in polysialogangliosides with increasing age. When isolated, dorsal root ganglia were incubated in the presence of a concentration of nerve growth factor (NGF) sufficient to promote the outgrowth of nerve fibres, there was increased incorporation of d -[1-¹⁴C]glucosamine into gangliosides. There was, however, no difference in the pattern of incorporation into gangliosides by control ganglia and those exposed to NGF.     

Specific binding sites for 125I-nerve growth factor in peripheral tissues and brain

Specific binding of ¹²⁵I-nerve growth factor (NGF), defined as that part of the total binding of the iodinated derivative displaced by 15–30 μg/ml native NGF, is found at significant levels in many peripheral tissues of chick embryos and rats. Destruction of the sympathetic innervation of tissues by treatment of newborn rats with guanethidine does not materially alter the ¹²⁵I-NGF specific binding capacity of tissues, indicating that these binding sites for NGF are part of the tissues themselves and not a property of the sympathetic nerve terminals which innervate them. Specific binding of ¹²⁵I-NGF which is also resistant to guanethidine treatment exists in chick embryonic and rat brain. The time course of the development of this specific binding in chick embryonic heart and brain suggests a developmental role for these peripheral and central nervous system NGF binding sites.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

The ontogeny of specific retrograde transport of nerve growth factor by motoneurons of the brainstem and spinal cord.

Radiolabeled Nerve Growth Factor (NGF) was injected into either the mandibular process of the first visceral arch or the limb bud of chick embryos at Days 3.5-14 or Days 4-13 of incubation, respectively. Control embryos received injections of labeled cytochrome-C or labeled NGF plus an excess of unlabeled NGF. The tissues were then processed for autoradiography. The 125I-NGF was retrogradely transported by motoneurons of the trigeminal (V) motor nucleus on Days 3.5-8 of incubation, but not at later stages. Similar transport was seen in motoneurons of the spinal cord lateral motor column from Days 4-10 of incubation, but not at later stages. Sensory neurons of the V ganglion and of the dorsal root ganglia transported NGF at all injection ages. In no instance was the 125I-cytochrome-C transported by sensory or motor neurons. The injection of an excess of cold NGF along with labeled NGF resulted in no evidence of retrograde transport of the labeled NGF indicating that the transport was saturable. The time of transport by these brainstem and spinal cord motoneurons corresponds closely to the points during development at which they have been found to exhibit specific NGF binding. The present results, then, provide further evidence for a possible biological role for NGF during early developmental stages of these motoneuron populations.     

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.