Ionic behaviors and nerve growth factor dependence in developing chick ganglia. II. Studies with neurons of dorsal root ganglia

Using intact dorsal root ganglia (DRG) from embryonic (E) chick and measuring 22Na+ accumulation, the authors have recently shown that (i) ionic control by the ganglia has a complete requirement for exogenous NGF between E6 and E10, and (ii) control of ion pump mechanisms independent of exogenous NGF is progressively acquired by these ganglia from E10 to E16. Similar experiments have now been carried out using enriched suspensions of ganglionic neurons to test whether the acquisition of endogenous control by older ganglia was (1) due to the close association between neurons and nonneurons, and (2) correlated with a decreasing need by these neurons for exogenous NGF for survival in culture. In this enriched neuronal population, Na+ accumulation in the absence of NGF increases from E7 to E10, paralleling the increase in Na+ accessible space under ouabain, but then decreases conspicuously between E10 and E16, despite little change in the ouabain-sensitive Na+ space. NGF prevents Na+ accumulation during the early period, and becomes increasingly irrelevant for this behavior in later (after E10) development. K+ movements (traced with 86Rb+) behaved similarly. Active K+ influx (Na+, K+-pump mediated) also increases severalfold between E7 and E10. This K+ influx is sensitive to NGF at E7 and E10 but not at E14, paralleling the observed Na+ and K+ behaviors. These data suggest that the control of Na+, K+-pump performances acquired by these neurons between E10 and E16 represents the development of a neuronal self-sufficiency. This increase in ionic control is not due to an increase in pump molecules or pumping efficiency. No increases in the binding of [3H]ouabain or active K+ influx occur between E10 and E16, when ionic control is developing. The ionic dependence on NGF by the DRG neurons changes with their developmental age along the same temporal pattern displayed by their survival response to NGF in culture.     

Short-latency ionic effects of nerve growth factor deprivation and readministration on ganglionic cells

Nerve growth factor (NGF) is likely to exert its trophic action on dorsal root ganglion (DRG) and on sympathetic ganglion neurons by controlling a crucial function of these cells. This function would in turn regulate other cellular machineries and, ultimately, lead to the traditional NGF consequences, such as survival and neuritic growth. A corollary of this view is that the key to NGF action must lie in short-latency events, occurring within minutes of NGF administration. Chick embryo DRG dissociates have proved to be an effective experimental system to investigate short-latency responses to NGF, in that (1) measurable functional deficits develop over 6 h of NGF deprivation in vitro and (2) delayed presentation of NGF promptly and fully restores the defective function. The first deficit observed in this experimental system, a decline in RNA-labeling capability, led to the recognition that NGF controls the transport of selected exogenous substrates, all of which are Na⁺-coupled and depend on an Na⁺ gradient across the neuronal membrane. Subsequent work showed that NGF controlled such transport systems by actually regulating the neuronal ability to control intracellular Na⁺. Under NGF deprivation, the DRG cells accumulate Na⁺ to levels that reflect, and presumably equate, the extracellular Na⁺ concentrations. Conversely, on delayed NGF administration, the accumulated Na⁺ is actively extruded to an extent and at a speed that depends on the NGF concentration. The Na⁺ response is elicited by both Beta and 7S NGF, but not by other proteins tested. All ganglionic systems that display a requirement for exogenous NGF in culture have also displayed the Na⁺ response to NGF. The Na⁺ response is grossly paralleled by a K⁺ response. DRG dissociates, in which intracellular K⁺ has been pre-equilibrated with extracellular ⁸⁶Rb⁺, lose their ⁸⁶Rb⁺ over 6 h of NGF deprivation and restore it on delayed NGF administration. The regulation by NGF of mechanisms controlling intracellular Na⁺ and K⁺ levels in their target neurons is likely to occupy an early and fundamentl place in the sequence of events underlying the mode of action of this factor.     

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     

Nerve Growth Factor influences potassium movements in chick embryo dorsal root ganglionic cells

Recently we have shown that Nerve Growth Factor (NGF) influences the movement of Na⁺ across the membrane of chick embryo dorsal root ganglion (DRG) cells. When cell dissociates from 8-day embryonic chick DRG, equilibrated with ⁸⁶Rb⁺ (a K⁺ analog) in the presence of NGF, were transferred to NGF-free medium a marked loss of intracellular K⁺ occurred over several hours. The time course of K⁺ loss was similar to the time course of Na⁺ accumulation which occurs in the absence of NGF. NGF-deprived, K⁺-depleted DRG cells reaccumulated K⁺ within minutes of delayed NGF presentation, just as delayed NGF administration results in the rapid extrusion of Na⁺ from Na⁺-loaded cells. Restoration of K⁺ competence was dependent upon NGF concentration. The occurrence of this K⁺ response to exogenous NGF in other ganglionic preparations correlated with traditional responses to NGF in culture and previously observed Na⁺ responses. Neither the development nor the expression of the ionic defect (K⁺ depletion, Na⁺ filling) during NGF deprivation required the presence of both cations in the medium. NGF-dependent restoration of intracellular K⁺ in NGF-deprived chick DRG cells required the presence of intracellular Na⁺, and NGF-dependent extrusion of Na⁺ required extracellular K⁺. Thus NGF appears to influence the coupled (active) movements of Na⁺ and K⁺ across the membrane of its target cells, possibly by means of the classical Na⁺, K⁺-ATPase pump.     

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.     

Both nerve growth factor and high K concentrations support the survival of chick embryo sympathetic neurons : Evidence for a common mechanism of action

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K⁺ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K⁺, and survival was not increased when both NGF and K⁺ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K⁺ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K⁺ and NGF for the survival of cultured embryonic neurons.     

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.     

Age-Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to 1-Methyl-4-Phenylpyridinium: Role of Glutathione

Abstract: We demonstrate that 1-methyl-4-phenylpyridinium (MPP⁺) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP⁺ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 µM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DMI; 10 µM for 30 min). MPP⁺ blocked the uptake of [³H]NE by sympathetic neurons in a dose-dependent manner with a potency roughly equal to DMI. At concentrations up to 10 µM, MPP⁺ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP⁺ diminished gradually with increasing lengths of time in culture. When MPP⁺ was added to the culture medium 48 h after plating, concentrations up to 100 µM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP⁺-induced cell death could not be explained by an increasing capacity for sequestration of MPP⁺ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP⁺ (1 µM) to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor l -buthionine-[S,R]-sulfoximine (1 µM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP⁺ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP⁺.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (～2 × 10^?11M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.     

Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons.

An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10(-9)-10(-8)M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: high-affinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation in the peripheral nervous system.     

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)     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons

Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (～2 × 10^?11M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.     

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.     

Pituitary Adenylyl Cyclase-Activating Polypeptide and Nerve Growth Factor Use the Proteasome to Rescue Nerve Growth Factor-Deprived Sympathetic Neurons Cultured From Chick Embryos

Abstract: Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24–48 h. Uptake of [³H]norepinephrine ([³H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1–100 nM) restored [³H]NE uptake to 92 ± 8% of that of NGF-supported controls. Depolarization-induced [³H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3′,5′-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 ± 40 versus 38 ± 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 ± 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.     

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.     

Rapid effects of nerve growth factor on the Na,K-pump in rat pheochromocytoma cells

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na⁺,K⁺-pump mediated K⁺ influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na⁺,K⁺-pump is completely blocked by the Na⁺-flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na⁺ ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na⁺ influx leading to an activation of the Na⁺,K⁺-pump, a mechanism similar to the action of other growth factors.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive ⁸⁶Rb uptake and amiloride-sensitive ²⁴Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na⁺/H⁺ and Na⁺/K⁺ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na⁺/H⁺ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na⁺/H⁺ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na⁺ pump, Na⁺/H⁺ exchange) without eliciting corresponding regulatory mechanisms (Na⁺ stat, pH stat).     

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.     

The thoracic sympathetic neurons of the chick: normal development and the effects of nerve growth factor

The generation and degeneration of sympathetic neurons in the third thoracic ganglion (segment 19) of the chick were studied between embryonic days (E) 7-18 using 3H-Thymidine autoradiography and routine cell counts. Cumulative radiolabelling experiments indicated that few sympathetic neurons were generated on E6-7. 10% of the sympathetic neurons were generated on E8 and a further 20% on E9. The final 70% of neurons completed the mitotic cycle between E10-12. Cell counts demonstrated that the neuronal population increased from 10,166 +/- 423 (mean +/- SEM) to 22,291 +/- 767 between E8-10 and remained stable up to E14. The population subsequently declined by 37%, to 14,157 +/- 831, by E18. Pyknotic neurons were found at all stages of development, but were most apparent between E7-15. The effects of Nerve Growth Factor (NGF) on the number of both surviving and pyknotic neurons in the ganglion were also examined. E9 embryos treated with NGF from E5-8 showed a 57% increase in the number of sympathetic neurons. This increase therefore occurred prior to the decline in neuronal number and was not accompanied by a decrease in the number of visibly pyknotic neurons. It is therefore possible that early NGF treatment increases the number of sympathetic neurons through a mechanism other than the attenuation of cell death.