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S D Skaper

Publications and source records attributed to S D Skaper.

At least 127 records · Page 7Linked to original sources
PubMed1985

Comparison of the effects of laminin and the polyornithine-binding neurite promoting factor from RN22 Schwannoma cells on neurite regeneration from cultured newborn and adult rat dorsal root ganglion neurons.

We have investigated the effects of two neurite promoting factors (NPFs)--laminin and the semipurified polyornithine-binding neurite promoting factor (PNPF-1) from RN 22 Schwannoma cells--on neurite regeneration from dissociated newborn and adult rat dorsal root ganglion (DRG) neurons during 24 and 48 h culture periods in the absence of exogenous neuronotrophic factors. Both laminin and PNPF, when used to pretreat the polyornithine substratum, significantly enhanced neurite recruitment from surviving newborn and adult DRG neurons as compared to an untreated polyornithine substratum. However, the responses of newborn neurons at saturating concentrations of laminin and PNPF were consistently greater (46% neurite-bearing cells at 24 h, 81% at 48 h) than those of adult neurons (14 and 45%, respectively). The responsive neurons of both newborn and adult DRG displayed extensive neuritic networks at 48 h. The ED50 of laminin, or PNPF was 0.15-0.2 micrograms/ml for both newborn and adult neurons. The similarities in the responses of newborn and adult DRG neurons to NPFs validate the use of neurons from embryonic and newborn animals for the in vitro assays of NPFs that can be collected from injured and regenerating adult peripheral nervous tissues.

Age Factors↗
PubMed1985

Specific replacements of pyruvate for trophic support of central and peripheral nervous system neurons.

When embryonic central nervous system neurons are seeded at low densities with Eagle's basal medium supplemented with the serum substitute N1, glucose, and glutamine, neuronal survival for even 24 h requires the additional supply of exogenous pyruvate--and so does the survival of many peripheral nervous system neurons. Pyruvate can be replaced by alpha-ketoglutarate or oxaloacetate, but not by Krebs cycle substrates that are not keto acids. Most other alpha-keto acids tested (though not beta- or gamma-keto acids) also mimic pyruvate. The apparent equivalence to pyruvate of all these compounds includes identical ED50 values (300 microM for embryonic avian fore-brain neurons, 30-40 microM for rat hippocampal neurons), and also identical susceptibilities to the pyruvate-sparing effects of other low-molecular-weight agents present in Dulbecco's modified Eagle's medium or in astroglia conditioned medium. The substitute alpha-keto acids, however--unlike pyruvate, alpha-ketoglutarate, or oxaloacetate--support cell survival only in the presence of alpha-amino acids that transaminate to alpha-ketoglutarate, oxaloacetate, or pyruvate. The alpha-keto acids, therefore, operate as acceptors of amino groups from appropriate donors to generate Krebs cycle-relevant substrates. Consistent with this view, [14C]glutamate did not generate appreciable 14CO2 unless accompanied by a suitable alpha-keto acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Pyruvate participation in the low molecular weight trophic activity for central nervous system neurons in glia-conditioned media.

Conditioned media from glial cell cultures contain low molecular weight agents which can support survival of CNS neurons in the absence of recognized protein neuronotrophic factors. A similar support is provided to CNS neurons by selected basal media, and pyruvate is the critical medium constituent responsible for their trophic competence. Eagle's basal medium, which contains no pyruvate, acquires pyruvate when conditioned over astroglial cell cultures. Enzymatic degradation of the pyruvate in the astroglia-conditioned medium leads to corresponding losses in its low molecular weight trophic activity for CNS neurons. Quantitative correlations between pyruvate content and CNS trophic activity demonstrate that pyruvate is the main trophic ingredient of the glia-conditioned medium, and other low molecular weight substances, acquired during conditioning, reduce the pyruvate concentration required for its trophic effect. The "pyruvate-sparing" substances, as yet unidentified, are not the serine and Fe3+ which have pyruvate-sparing competence for peripheral, ciliary ganglionic neurons. These findings, together with previous observations, propose that prenatal neurons fail to generate or retain endogenous pyruvate at the levels for their survival-sustaining activities.

Animals↗
PubMed1984

Chemically defined requirements for the survival of cultured 8-day chick embryo ciliary ganglion neurons.

We have previously demonstrated that both peripheral and central neurons from embryonic chick and newborn mouse can be maintained in a serum-free defined culture medium containing the appropriate neuronotrophic agent and the N1 supplement consisting of insulin, transferrin, putrescine, progesterone and selenite. In the present studies we have examined the short-term survival requirements of 8-day embryonic chick ciliary ganglion (CG) neurons. By comparing CG neuronal survival in our standard culture medium, Eagle's Basal Medium (EBM), with several other commercially available basal media, we have established that CG neurons also have specific requirements for pyruvate, serine and iron (Fe3+), in addition to their trophic factor (Ciliary Neuronotrophic Factor, CNTF) and the N1 supplement. The data suggest the existence of 3 subsets of CG neurons differing in their essential needs, namely: (1) those supported by glucose in the absence of pyruvate, (2) those requiring exogenous pyruvate but not serine or Fe3+, and (3) those which need pyruvate, serine and Fe3+. The minimal effective concentration of pyruvate could be decreased by a factor of 50 in the concurrent presence of serine and Fe3+. Serine was also a limiting element in the survival of some of these CG neurons. The Fe3+ concentration required by the same neurons was considerably diminished with the availability of transferrin, perhaps reflecting an increased Fe3+ transmembrane transport efficiency. Insulin was found to be the only N1 ingredient required for the survival of CG neurons. Insulin was a constant requirement for all 3 subsets of CG neurons, even when cultured in the total absence of glucose (but presence of pyruvate).

Animals↗
PubMed1984

Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro.

Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later.

Animals↗
PubMed1984

Interaction of GM1 ganglioside with PC12 pheochromocytoma cells: serum- and NGF-dependent effects on neuritic growth (and proliferation).

The effects of ganglioside GM1 on proliferation and neuritic growth of PC12 pheochromocytoma cells were studied in the presence and absence of nerve growth factor (NGF). In the absence of NGF, but not in its presence, a decrease in the total number of PC12 cells was first observed after 4-6 days of culture with 10(-6) M GM1 in 0.1% fetal calf serum, and with 10(-3) M GM1 on 10% serum. NGF, with or without GM1, limits cell growth to the first 4-6 days. GM1 enhanced neuritic recruitment with serum concentrations of 0.3% or more. Optimal neurite response varied from 10(-6) M GM1 with 0.3% serum to 10(-4) M GM1 with 10% serum. The influence of GM1 on neurites became more pronounced with increasing serum concentrations, becoming maximal with 1% or greater serum. Serum exhibited a concentration-dependent inhibitory influence (lag) on NGF-induced neuritic recruitment, which was abolished by GM1. Rates of neuritic recruitment following the lag were unaffected by GM1, while showing an inverse correlation with serum concentrations of 0.1-0.5%. Serum may delay the NGF-induced neuritic recruitment of PC12 cells by two independent mechanisms. These results suggest that GM1, in some manner, prevents the serum-induced delay in the onset of neuritic recruitment, rather than stimulating the rate at which it precedes.

Cell Differentiation↗
PubMed1984

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)

Animals↗
PubMed1984

Low molecular weight agents support survival of cultured neurons from the central nervous system.

Microcultures of dissociated neurons from various central and peripheral neural tissues were used for quantitative analysis and characterization of trophic agents directed to these test neurons. Media conditioned by a variety of central and peripheral glial as well as muscle cell cultures contain trophic activities for central neurons which are distinct from the more traditional protein factors directed to peripheral neurons, by at least two features: (1) they reside with low molecular weight (Mr less than 1000) agents which are resistant to heat, extremes of pH, and various proteolytic and peptidolytic enzymes; and (2) they are necessary for the short-term survival of a variety of rodent and avian central neurons, but they fail by themselves to support survival of peripheral neurons under the same culture conditions.

Animals↗
PubMed1983

Neuronotrophic activity in brain wounds of the developing rat. Correlation with implant survival in the wound cavity.

Neuronotrophic activity accumulates in a wound cavity created in the entorhinal/occipital cortex of developing rats. These trophic factors support the survival of neurons in monolayer cultures of chick embryo spinal cord, ciliary ganglion, sympathetic ganglion and dorsal root ganglion, as well as of mouse dorsal root ganglion. Trophic activity was very low both in non-injured brain tissue and in the wound cavity 1 day post-lesion, but it increased 15- to 300-fold during the subsequent 2-5 days. Together with the trophic activity in the wound fluid were other substances which interfered with the survival of spinal cord neurons. The neuronotrophic factors appeared to be proteins immunologically distinct from mouse submaxillary nerve growth factor. Fragments of rat embryo corpus striatum placed in the cortical wound cavity immediately after its formation showed very poor subsequent survival and no innervation of the host hippocampus. However, if implantation was delayed by 3 or 6 days with respect to the time at which the receiving cavity was made, the survival was greatly improved and innervation of the host took place. The time course for the accumulation of the trophic factors in the cavity paralleled the delay leading to increased survival of brain grafts. It is suggested that the neuronotrophic activity accumulating in the wound cavity during the delay period may be responsible for the increased survival of the implants.

Animals↗
PubMed1983

Ionic behaviors and neuronal survival in developing ganglia. III. Studies with embryonic chick sympathetic neurons.

We have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na+, K+-pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na+ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13-E18), NGF becomes less and less required for that control as the neurons gain a self-sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.

Animals↗
PubMed1983

Serum- and substratum-dependent modulation of neuritic growth.

Explants of embryonic day 8 (E8) chicken dorsal root ganglia (DRG) have been cultured with medium containing serum or the serum-free supplement N1 on one of three substrata: collagen, polyornithine (PORN), or PORN exposed to a polyornithine-binding neurite-promoting factor (PNPF-PORN). Replicate cultures were maintained with or without nerve growth factor (NGF). NGF elicited its classical neuritic outgrowth on all three substrata in serum-containing or serum-free medium. In the absence of NGF, however, a gradation of increasing neurite growth was seen with: PNPF-PORN greater than PORN greater than collagen. This response occurred in both media. In addition, the neuritic halo in each instance was markedly more developed in the absence of serum, especially on PNPF-PORN. Nonneuronal behaviors reflected both serum and substratum influences: thus, nonneuronal outgrowth consisted mainly of flat cells with serum and collagen, was nonexistent with serum and PORN or PNPF-PORN, and involved mostly Schwann-like scattered cells in the absence of serum on any one substratum. The serum-dependent behaviors of ganglionic neurites were examined further with explants from chicken E11 sympathetic ganglia. A single substratum was used (PORN), without exogenous trophic factor. Neurite outgrowth was depressed by the presence of fetal calf serum, thus supporting the generality of this phenomenon. Lastly, PC12 cells, a clonal line of rat pheochromocytoma, will grow neurites in the presence of NGF after 48 hr in serum-free, but not serum-containing media. Addition of serum to serum-free cultures at this time results in the rapid and complete retraction of neurites.

Animals↗
PubMed1983

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells.

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.

Blood Proteins↗
PubMed1983

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.

Age Factors↗
PubMed1983

Temporal changes of neuronotrophic activities accumulating in vivo within nerve regeneration chambers.

The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.

Animals↗
PubMed1983

Age-dependent requirements of sympathetic neurons in serum-free culture.

Neurons dissociated from chick sympathetic ganglia of different embryonic ages can be cultured equally well in serum containing or serum-free (N1) medium on collagen polyornithine, and polyornithine pretreated with neurite-promoting factor (PNPF), in the presence of nerve growth factor (NGF). Quantitative analysis of nerve cell survival is best achieved in serum-free conditions and on PNPF-pretreated polyornithine. Under such conditions, 11-day embryonic (E11) sympathetic neurons showed a specific requirement for insulin, selenium and transferrin. Similar analyses at E8 and E16 revealed that the requirement for transferrin is developmentally regulated since it is not expressed by E8 nerve cells until after 3 days in vitro. These data strongly suggest that, besides NGF, other specific molecules are involved in the control of neuronal survival and that specific requirements may be displayed at different embryonic stages.

Animals↗