摘要:

AbstractSurvival in monolayer culture of 4‐day (stage 23) chick embryo lumbar spinal cord neurons can be regulated by two opposing activities. One, spinal neuronotrophic activity, promotes neuronal survival; and the other, spinal neuronotoxic activity, eliminates the neurons from the culture even when the trophic support is present at an optimal concentration. Quantitative microbioassays for each activity are presented and used to measure the relative amounts of each agent within different sources including glial, muscle, and spinal cord cell‐conditioned media and fluid collected from peripheral and central nervous tissue lesions. Although both activities were present in all of the sources tested, their concentrations in the wound fluids were orders of magnitude greater than in the conditioned media. The fluid‐derived trophic activities were inactivated by heat and trypsin and nondialyzable, whereas all of the conditioned media‐derived trophic activities were heat‐ and trypsin‐resistant and

ISSN:0360-4012    

DOI:10.1002/jnr.490080214

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractWe have previously demonstrated that both peripheral and central neurons from 8 day embryonic chick and newborn mouse can be maintained in a serum‐free medium using the N1 supplement consisting of insulin, transferrin, putrescine, progesterone, and selenite. In the present studies we show that dorsal root ganglionic (DRG) neurons from embryonic chick (E7–E15) and neonatal mouse can be cultured in a serum‐free environment with only the addition of insulin and transferrin, plus Nerve Growth Factor (NGF). Chick DRG from E10–E15 contain a population of neurons sensitive to a chick embryo eye‐derived ganglionic neuronotrophic factor (GNTF), which is distinct from the neuronal subset dependent upon NGF. The GNTF‐dependent chick neurons can also be maintained in culture with insulin and transferrin supplements. Neonatal mouse DRG neurons, whether supported by NGF or eye‐derived GNTF, likewise survive in serum‐free medium with only insulin and transferrin. Limited numbers of neurons survive for the first 24 hours in a serum‐free medium lacking insulin or transferrin, but failed to display neurite outgrowth even in the presence of add

ISSN:0360-4012    

DOI:10.1002/jnr.490080215

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractCulture of embryonic chicken dorsal root ganglia for periods exceeding a week generally require serum supplementation and a trophic factor such as nerve growth factor. In this communication we describe the culture of chicken E‐9 dorsal root ganglia for period up to 2 weeks in a system that is composed only of a simple basic salts solution and 10 ng/ml (3.8 × 10−10M) β nerve growth factor. Commercially available tissue culture dishes are used which have a hydrophilic, gas‐diffusable membrane on the bottom to which the ganglia directly attach, eliminating the need for added substratum. Sparse fiber outgrowth appears after 24 hours and growth of the fibers continues for 120 hours of incubation. At this time, the fibers are extremely dense and reach approximately 3.5–4.0 diameters from the body of the ganglion. Continued survival of these fibers appears to depend on the concentration of nonneuronal cells present in the dish. No fiber outgrowth occurs at any time in the absence of β nerve growth factor. The simplicity and purity of this culture system makes it an attractive tool in the study of primary cell‐cell interactions, the production of trophic factors by non‐neuronal cells, and biochemical and physiological analyses of g

ISSN:0360-4012    

DOI:10.1002/jnr.490080216

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractConditioned medium (CM) from muscle or fibroblast cultures dramatically increases the outgrowth of neurites from fetal rat spinal cord slices in vitro. We report here that there are two separable fractions in conditioned medium that cause this increase in neuritic outgrowth. One fraction, with a molecular weight of approximately 50,000 daltons, enhances neuritic outgrowth only when it is present in the culture medium so that the slices are directly exposed to it. The second component has a much larger molecular weight (above 300,000 daltons), and can enhance neuritic outgrowth by directly binding to the culture substrate on pretreatment. Only when these two fractions are combined by pretreating the substrate with the higher molecular weight fraction and then growing the slices in the lower molecular weight fraction is the full outgrowth promoting activity of whole conditioned medium reconstituted. These two components act synergistically to reproduce nearly the full outgrowth promoting activity of non‐fractionated, whole conditioned mediu

ISSN:0360-4012    

DOI:10.1002/jnr.490080217

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractSeven day chick embryo cerebral cortical neurons cultured at low density (10,000 cells/16 mm well) in serum‐free, defined medium in polylysine‐coated wells fail to extend neurites and assume a flattened phase‐dark morphology. Addition of serumfree, defined medium conditioned over chick embryo heart cells promotes neurite outgrowth and rounding of the cell body (which becomes phase bright). A sensitive bioassay, based on counting the number of phase‐bright neurons with processes at least equal to one cell body diameter after 20 hours in culture, was used to titrate neurite‐promoting activity in heart conditioned medium. This activity is completely destroyed by exposure to trypsin. As little as 2 μg/ml total conditioned medium protein elicits a half‐maximal response in t

ISSN:0360-4012    

DOI:10.1002/jnr.490080218

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractA new neuronal growth factor (CMF) isolated from mouse heart‐cell‐conditioned medium stimulates morphologic and biochemical development of mouse embryo sympathetic neurons [Coughlin et al, 1981]. Further analysis of CMF by chromatographic and electrophoretic procedures has shown that under nondissociating conditions, CMF activity is associated with very high molecular weight material. All biological activity eluted within the included volume of a Sepharose CL‐2B column in a molecular weight range corresponding to approximately 5 × 106daltons. Similarly, electrophoresis showed no activity and very little protein entering 3% polyacrylamide gels, whereas both protein and activity migrated through 0.6% agarose‐1.2% acrylamide composite gels. To further characterize the biological effects of CMF on normal neuronal development, antibodies to CMF were employed. Rabbits immunized against CMF developed high titres of antibodies with activity specifically directed against CMF (anti‐CMF). Although anti‐CMF inhibited nerve growth factor (NGF)‐stimulated neurite outgrowth from the neonatal superior cervical ganglion, it did not inhibit the NGF‐stimulated increase in tyrosine hydroxylase activity. Moreover, ganglia incubated for 3 days in the presence of anti‐CMF were subsequently capable of producing neurites when washed and cultured in medium free of antiserum. Thus, anti‐CMF specifically blocked neurite extension without causing cell death or irreversible damage in ganglionic explants. Our observations suggest, therefore, that CMF or antigenically similar material is a requirement f

ISSN:0360-4012    

DOI:10.1002/jnr.490080219

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractChick neuroblasts from 6‐day‐old embryos were grown on a collagen substrate in minimum Eagle's medium supplemented with 5% horse serum. We found that [3H]thymidine incorporation in these cultures decreased drastically from day 2 to day 3 after seeding. Addition of brain extract from 8‐day‐old chick embryos (CBe8) to the nutrient medium after 24 hours of culture elicited a very important stimulation of [3H]thymidine incorporation. Extracts of brains from adult animals were much less effective. It was shown that the increase of tritiated‐thymidine incorporation reflected proliferation of neuroblasts in the culture, at least between day 2 and 4. The activity of the brain extracts was destroyed by trypsin treatment, which suggests that the factor responsible for the stimulation of the proliferation is a polypeptide. The CBe8 and the brain extract of adult chicken (CBa) were fractionated by gel filtration. An active fraction was eluted at a volume indicating an apparent molecular weight of roughly 70,000. At that stage of the study results suggest that a polypeptide (tentatively called neuroblast proliferation factor, NPF) present in the embryonic chick brain extract, is a proliferation factor for chick neuroblasts in primar

ISSN:0360-4012    

DOI:10.1002/jnr.490080220

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe minimal requirements for the regeneration of optic nerve fibers in vitro were established in a serum‐free retinal explant preparation. This serum‐free preparation was developed as a prerequisite for testing the growth‐promoting activity of tissue extracts prepared from the primary target of regenerating fibers. Explants taken from goldfish retinas 14 days after a prior optic nerve crush were capable of long‐term survival and regenerated neurite outgrowth without serum or hormonal supplements. Serum‐free conditions for explant outgrowth required only a basic Leibovitz (L‐15) media containing 0.6% methyl cellulose (MC). Explants were also capable of neurite outgrowth in L‐15 media alone when culture dishes were preplated with MC. MC treatment permitted both the regeneration of neurites in serum‐free L‐15 and a significant increase in the rate and extent of neurite outgrowth when combined with 10% fetal calf serum (FCS). Explants grown in L‐15 with both MC and FCS produced a 2.5‐fold increase in the length of neurite outgrowth over MC alone and a 1.5‐fold increase in the length of neurite outgrowth over FCS alone. MC activity which permitted minimal serum‐free regeneration and optimal serum supplemented regeneration was determined to be substrate related. Retinas were dissociated to determine if ganglion cells, like the intact explant, were capable of survival and neurite regeneration in serum‐free conditions. These cells survived and extended long neurites when grown in L‐15 with FCS or with FCS and MC, but they did not survive in serum‐free L‐15 with MC.The minimal serum‐free conditions for explant for explant survival and neurite regeneration were used as a model system to test the growth‐promoting activity of crude tissue extracts prepared from the goldfish brain. Extracts prepared from the primary target region, the optic tectum, stimulated a significant 2.5‐fold increase in the length of regenerating neurites. The optic tectal extract (OTex) stimulated outgrowth with significantly high specific activity when compared with extracts of identical protein concentrations prepared from the cerebellum (Cex). At a minimal protein concentration of 150 μg/ml, the OTex stimulated a 1.5‐fold increase in neurite outgrowth above Cex.These results indicated that a serum‐free culture preparation had been established for optic nerve regeneration. This culture system has proven to be an extremely sensitive bioassay model without the masking effect of a serum supplement. Serum‐free cultures may be used in further studies to determine the role neurotrophic factors may play in a widely used model of succes

ISSN:0360-4012    

DOI:10.1002/jnr.490080221

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractTwo proteins (ependymins β and γ) localized within specific cells in the zona ependyma of goldfish brain were identified in previous studies in our laboratory as extracellular factors whose metabolism was highly increased by the acquisition of new patterns of behavior. Methods for growing zona ependyma cells in culture were developed to determine if cells within this tissue could synthesize and release the ependymins into their extracellular environment. Dissociated cells from zona ependyma were grown as primary monolayer cultures on polylysine‐coated slides. The cultures survived for at least 4–5 weeks, forming an intricate network of interconnecting processes. These contained many cell types, several of which retained their in vivo immunohistochemical properties. Specific populations of cells staining with antisera to nerve growth factor, glial fibrillary acidic protein, and ependymins β and γ were present. Radioimmunossay, immunoelectrophoresis data, and labeling experiments indicate that cells within the cultures can synthesize the ependymins de novo and release them into their extracellular environment. These results are consistent with the hypothesis that the ependymins might function as extracellular factors which mediate the behaviorally induced plasticity of the goldfish CNS. In this respect they resemble other “protein factors” which stimulate the growth and morphological differentiation of nervous syst

ISSN:0360-4012    

DOI:10.1002/jnr.490080222

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe effects of target tissues on the neural centers of the developing spinal reflex arc of frog tadpoles have been integrated to form a model explaining the spatial and temporal interactions of neuron and target. Spinal cord responds to appropriate targets earlier than does sensory ganglion in tissue culture. Neuronal growth and survival responses to target tissues in vitro may explain the regulation of development in situ.

ISSN:0360-4012    

DOI:10.1002/jnr.490080223

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY