L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC12 pheochromocytoma cells

Two mouse cell lines, 3T3 and L, reported to secrete an NGF-like activity in the culture medium were co-cultured with the pheochromocytoma cell line PC12 which responds to NGF in vitro. In these co-cultures mitomycin-treated L or 3T3 cells were employed at low cell density (1 000 cells/cm2). L cells, but not 3T3, promoted efficiently neurite outgrowth of PC12. The response of the PC12 cells was blocked by an antiserum to male mouse submaxillary gland beta NGF. The NGF secreted by the L cells and immunoprecipitated by this antiserum co-migrated with the submaxillary gland beta NGF monomer in SDS-polyacrylamide gels. Surprisingly the neurite-promoting activity of media conditioned by L or by L-PC12 co-cultures was at most one-tenth of that expected on the basis of the response of PC12 cells in the co-cultures. This was not due to proteolytic degradation of the NGF-like factor or to losses by manipulation of the media. It seems therefore that co-cultures provide conditions which enhance the effect of the factor. Possible mechanisms responsible for this effect are discussed.     

Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Nerve Growth Factor Mediates Monosialoganglioside-Induced Release of Fibronectin and J1/Tenascin from C6 Glioma Cells

C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.     

Nerve growth factor (NGF) loop 4 dimeric mimetics activate ERK and AKT and promote NGF-like neurotrophic effects

Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite- but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.     

Guaiacol and vanilloid compounds modulate the A-type potassium currents in molluscan neurons

The actions of guaiacol (2-methoxy-phenol), vanillin (4-hydroxy-3-methoxy-benzaldehyd) and other vanilloid compounds such as zingerone (4-/4-hydroxy-3-methoxyphenyl/-2-butanon) and eugenol(2-methoxy4-/2-propenyl/phenol) were investigated on the fast outward potassium currents (A-type currents) in molluscan neurons. Guaiacol (0.01-0.1%, w/v) moderately decreased the peak amplitude but increased the rate of inactivation of the A-currents in dose-dependent way (Kd = 0.06% 4 mM, nH = 0.8). Vanillin (5 mM) slightly decreased the peak amplitude of the A-currents in Helix neurons but its action was more pronounced in dialysed Lymnaea nerve cells. However, vanillin similarly decreased the time-to-peak and the time constant of decay of the A-currents both in the faster and the slower inactivating Lymnaea and Helix neurons (Kd = 5 mM, nH = 0.6). The voltage-dependence of activation and inactivation of the A-currents were not significantly influenced by guaiacol and vanillin in Helix or Lymnaea neurons. Vanillin hardly influenced the delayed outward currents, but decreased the leak currents in the identified LPa and RPa 2,3 neurons. A structure-activity analysis clearly showed that increasing alkyl tail length from the aldehyde side of the vanillin molecule increased the efficacy of the various compounds on the amplitude of the A-currents and modified the kinetical influence on the A-current channel. Furthermore, an attenuation of the late outward currents and an increase of the leak conductance also developed in the presence of zingerone or eugenol. Excitatory actions of the studied vanilloids predominated on the various molluscan neurons.     

Induction of fibre outgrowth and choline acetyltransferase in PC12 pheochromocytoma cells by conditioned media from glial cells and organ extracts

Culture medium conditioned by C-6 glioma cells (previously shown to support the survival of chick sensory neurons in vitro [7]) was tested on the PC12 clone of pheochromocytoma cells. It is demonstrated that fibre outgrowth can be induced and the activity of the enzyme choline acetyltransferase stimulated by this medium. The activity of the conditioned medium is protease sensitive and the peak activity has an apparent molecular weight (on gel filtration) of approx. 50000. Similar effects can be obtained by conditioned medium from another rat glioma, D-74, and by extracts from rat central and peripheral nervous system. In no case is the effect blocked by specific antibodies against nerve growth factor (NGF). Media conditioned by other cell lines, together with other tissue extracts (at the same concentration as those taken from nervous tissue) have no detectable effect on the PC12 cells. It is suggested that PC12 cells will be useful as a tool for the further characterisation of factors with NGF-like activities.     

Neurotrophic actions initiated by proNGF in adult sensory neurons may require peri-somatic glia to drive local cleavage to NGF

The nerve growth factor (NGF) precursor, proNGF, is implicated in various neuropathological states. ProNGF signals apoptosis by forming a complex with the receptors p75 and sortilin, however, it can also induce neurite growth, proposed to be mediated by the receptor of mature NGF, tyrosine kinase receptor A (TrkA). The way in which these dual effects occur in adult neurons is unclear. We investigated the neurotrophic effects of proNGF on peptidergic sensory neurons isolated from adult mouse dorsal root ganglia and found that proNGF stimulated neurite extension and branching, requiring p75, sortilin and TrkA. Neurite growth rarely occurred in sortilin-expressing neurons but was commonly observed in TrkA-positive, sortilin-negative neurons that associated closely with sortilin-positive glia. ProNGF was unable to induce local trophic effects at growth cones where sortilin-positive glia was absent. We propose that in adult sensory neurons the neurotrophic response to proNGF is mediated by NGF and TrkA, and that peri-somatic glia may participate in sortilin- and p-75 dependent cleavage of proNGF. The potential ability of local glial cells to provide a targeted supply of NGF may provide an important way to promote trophic (rather than apoptotic) outcomes under conditions where regeneration or sprouting is required.     

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey,Lampetra japonica

Summary In the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.     

Presence of a nerve growth factor-like immunoreactivity in auditory receptors during postnatal development in the rat

The presence of nerve growth factor (NGF) was investigated in the rat cochlea from birth to the adult stage, using immunohistochemical techniques NGF-like protein could be detected in the organ of Corti from birth up to day 8 and located within the hair cells, above the nuclei. No NGF-like immunoreactivity could be detected in the spiral ganglion. These results suggest that NGF may have a neurotropic action in the developing rat cochlea.     

Merkel cells in vitro: production of nerve growth factor and selective interactions with sensory neurons

A method has been developed for obtaining mixed primary cultures of dissociated epidermis enriched in Merkel cells. Merkel cells obtained from embryonic rat buccal pads were grown in serum-free medium and identified in vitro using a variety of histological and immunohistochemical markers. Quinacrine, a fluorescent amine, which has been used to identify Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. Cells labeled with quinacrine had a large, phase bright nucleus with prominent nucleoli, surrounded by a phase dark perinuclear ring. Antibodies directed against neuron-specific enolase, another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of quinacrine fluorescent cells. Electron microscopic examination of our cultures indicated that cells containing characteristic features of Merkel cells including cytoplasmic dense-cored granules were present. A small but significant increase in the number of Merkel cells was observed over time in culture. Merkel cells supported the survival and outgrowth of both trigeminal ganglion sensory neurons and sympathetic neurons from the superior cervical ganglion in serum-free medium in the absence of exogenous nerve growth factor (NGF). Immunoblots probed with antibodies directed against NGF demonstrated that NGF was present in the medium taken from these cultures. NGF-like immunoreactivity colocalized to cells containing quinacrine fluorescence in situ and in vitro. Addition of antibodies directed against NGF to cocultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and decreased survival of sensory neurons by 60%. These results suggest that Merkel cells are capable of providing trophic support for their normal complement of sensory neurons by producing NGF. Selective recognition of these targets was studied in vitro by characterizing the interactions between Merkel cells and growth cones from sensory or sympathetic neurons using both time-lapse videomicroscopy and standard morphometry of fixed cocultures. The majority of trigeminal ganglion sensory neurons (approximately 60%) extended growth cones onto clusters of Merkel cells. Neurites which contacted clusters of Merkel cells were significantly more highly branched than those growing on collagen. In contrast, the majority of sympathetic neurons (greater than 90%) failed to grow onto Merkel cells. Growth cones of sympathetic neurons often "collapsed" and retracted when contact was made with a cluster of Merkel cells. Fixation of Merkel cells with paraformaldehyde prior to coculture did not affect this difference between sensory and sympathetic neurite extension onto the Merkel cells. However, prior fixation of Merkel cells eradicated the apparent Merkel ce-induced branching of sensory neurites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

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     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.     

The role of central aminergic neurons in the action of 20-hydroxyecdysone on neurosecretory cells of Rhodnius prolixus

The enhancement of electrical activity of the neurosecretory cells in the brain and corpus cardiacum of Rhodnius prolixus induced by 20-hydroxyecdysone has been used as a means of examining the role of aminergic neurons in this reflex. The response of the brain and corpus cardiacum from mated ovariectomized females to 20-hydroxyecdysone was blocked by phentolamine and phenoxybenzamine (α-aminergic receptor antagonists) but not by propranolol (a β-aminergic receptor antagonist). Preparations taken from ‘reserpinzed’ females failed to respond to 20-hydroxyecdysone. Dopamine at 10^?7 M was capable of mimicking 20-hydroxyecdysone in activating the neurosecretory system from mated ovariectomised females as well as from ‘reserpinized’ mated ovariectomised females. The response to dopamine was blocked by phentolamine. The neurosecretory system from virgin ovariectomized females failed to respond to 10^?7 or 10^?6 M dopamine, but was activated by 10^?5 M dopamine.It is concluded that the action of 20-hydroxyecdysone onto the neurosecretory cells is indirect and involves aminergic interneurons. The results also suggest that the mating stimuli may function by enhancing the response of neurons to amines.     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.     

Effect of the endogenous peptide factor and vasopressin on the neurons of mollusks of different ages

Electric reactions of the identified neurons in adult (10-12 months) and old (22-24 months) molluscs Lymnaea stagnalis to endogenous peptide isolated from the water soluble fraction of the Helix Pomatia brain homogenate as well as to vasopressin are studied. The endogenous peptide causes a significant activation of the burst both in adult and old neurons of the molluscs. No significant age-related differences in the pronouncement of the above shifts of the molluscan neurons are found. There is an increased sensitivity to the identified peptide, vasopressin, in the old individuals. A non-uniform change in the sensitivity of mollusk neurons to various neuropeptides may affect the synaptic conduction in old age.     

Fibronectin-like immunoreactivity in Helisoma buccal ganglia: evidence that an endogenous fibronectin-like molecule promotes neurite outgrowth

We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.     

Neutrophic influence of denervated sciatic nerve of on adult dorsal root ganglion neurons

Isolated adult frog dorsal root ganglion neurons survive in vitro in a defined medium for more than 4 weeks and extend processes. When co-cultured with a 1-mm piece of peripheral nerve the average tottal process lenght per neuron was 10 times longer than that of control neurons by 8 days, and the processes had a significantly different morphology from that of control neurons. This influence on process length increased with increasing time of nerve denervation length increased with increasing time of nerve denervation prior to co-culturing. These results suggest the release of a neurotrophic factor/s from the cells of the peripheral nerve. The neurotropic influence was completely blocked by antibodies against mouse nerve growth factor (NGF). Although NGF increased the average process length by twofold over control neurons, its influence never reached that of the nerve-released factor, and the NGF-induced processes had a distinctly different morphology. The frog nerve-released factor promoted process outgrowth from E11 chick sympathetic ganglia, although the process number, length, and their fasciculation differed greatly from those induced by NGF. These results suggest that the nerve-released factor/s are immunologically and functionally related to NGF but have not estabished whether a single factor or an aggregate of several secreted molecules are responsible. This article presents a new preparation in which the varied influences of different neurotrophic factors can be studied in great detail on large populations of isolated adult vertebrte neurons and sets the stage for the characterization and isolation of the frog peripheral nerve neurotrophic factor, as well as examining the influence of this facor on neuronal morphology and its ability to direct process outgrowth. 1994 John Wiley & Sons, Inc.     

Sympathetic neuronal survival induced by retinal trophic factors

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti‐NGF (1 μg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1–50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin‐receptors TrkA, TrkB, or TrkC had no effect on RCM‐induced sympathetic neuron survival. The survival effects were not blocked by anti‐GDNF, anti‐TGFβ, and anti‐CNTF and were not mimicked by FGFb (0.1–10 nM). LY294002 at 50 μM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high‐molecular weight retinal secreted factor that supports sympathetic neurons in culture. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 13–23, 2002