Cell surface modifications in neuronal maturation

Proceedings of the National Academy of Sciences, 1980

We report a simplified method for culturing fetal central nervous system cells predominantly inducing neurons that grow, differentiate, and live in vitro for as long as 10 weeks. These central nervous system cells form a confluent cell culture in which about 80% of the cells are fully differentiated neurons producing interconnecting axons and dendrite processes and live upon a sparse underlying population of fibrillary and protoplasmic astrocytes, oligodendrocytes, and fibroblasts. Morphological and cytochemical characteristics of these cell types, based on immunofluorescent cell specific markers and silver staining of neurons, are presented.

International Journal of Developmental Neuroscience, 1995

Differentiation, 1977

Neuroblastoma cells, grown in monolayer, transform, emit cytoplasmic processes, and acquire morphological and functional properties resembling those of mature neurons, whereas in suspension culture they remain in the undifferentiated anaplastic form. The appearance of intermediate (10 nm) filamentous structures in neuroblastoma cells is generally considered to indicate a state of cellular differentiation, one of a progressive sequence of maturing phases which lead the cell to the final differentiated state.We have examined by electron microscope murine C 1300 neuroblastoma cloned cells, grown in suspension or in monolayer cultures in the presence or absence of BrdU as an inducing agent and have compared the expression of intermediate filaments. These filaments were present in five clones of cells grown in suspension still in undifferentiated anaplastic form. One clone in particular showed a massive expression of filaments, particularly visible in the perinuclear region. One hundred per cent of the cells observed presented filaments whose number apparently increased when cells were grown in the presence of BrdU in suspension or in monolayer. One clone never showed intermediate filaments under any circumstances. The original line from which clones were derived showed poor expression of filaments which were visible only in cells grown in monolayer. These results suggest that the expression of intermediate filaments in neuroblastoma cells should be viewed as the result of a positive genetic control of phenotype expression rather than the result of a progressive sequence of differentiating events.

Experimental Cell Research, 1981

The distribution of several structural proteins in the extending neurites and growth cones of cultured embryonic mouse spinal cord neurons was studied by indirect immunofluorescence, using affinity chromatography-purified antibodies. Fibroblastic cells in the same cultures served as internal standards for the evaluation of staining intensities, Anti-tubulin, anti-actin, and anticlathrin stained neurons and their processes intensely, while staining with anti-ol-actinin was only moderate compared with fibroblasts. Microtubules were resolved by anti-tubulin in the 'palm' of the growth cone but not in the neurite. Anti-actin stained even the finest lamellae and tilopodia of the growth cone, and the neurite. Anti-cr-actinin revealed an irregularly speckled pattern of cross-reactive material in the neurite and in the palm of the growth cone and was absent from the tilopodia. Anti-clathrin stained the neurite intensely and homogeneously, and to a lesser extent the palm of the growth cone. The staining with antibodies against tubulin and clathrin differed grossly between neurons and fibroblastic cells. Within the neuron there were only gradual differences in staining intensities. The growth cone was not qualitatively different from the rest of the neurite, except for the tilopodia which lacked tubulin and a-actinin, similar to the microvilli of epithelial cells.

Developmental Brain Research, 1984

Dissociated chick embryo peripheral and central nervous system cultures enriched for neurons by differential adherence, defined medium and cytosine arabinoside release to the culture environment molecular species which enhance the performance of neurons in limiting conditions. Culture medium conditioned by the neurons can be depleted of substrate-attached material by serial passage on poly-o-lysine substrate, leaving in the medium factors which promote neurite outgrowth on poly-o-lysine. The substrate-attached material which enhances neuron survival and neurite extension is heat-and trypsin-labile but is not affected by prior treatment with antisera to mouse NGF, human plasma fibronectin or laminin. The autostimulation phenotype displayed by neurons may play a role in neuronal survival or axonal growth during neuronal development.

Brain Research, 1986

A tissue culture model has been developed to examine the hypothesis that proliferating non-neuronal cells may constitute a physical and/or chemical barrier to regenerating neurons in the central nervous system. Explants from the sensorimotor cortex of 20-day-old fetal rats were cultured in serum medium (control) or serum medium containing 10-5 M cytosine arabinoside (AraC), a mitotic inhibitor, for varying periods: 2-10, 4-12, 4-10, 4-8 and 4-7 days in vitro (DIV). The center and outgrowth zone of the explants were examined by phase-contrast microscopy at varying intervals between 3 and 18 DIV. The extent of central degeneration was greatest in explants treated with AraC from 2 DIV, and was least in the 4-7 day treated group in which only minimal degeneration was evident at 13 and 18 DIV. In the outgrowth zone at 18 DIV non-neuronal cell proliferation was controlled in the 4-10 day treated explants, although this was accompanied by extensive degeneration of neurites. Further examination of neurite viability, using a neurite viability ratio, revealed that degeneration was first evident at 6 DIV in the 2-10 day treated explants, but not until 9 or 13 DIV in any of the explants exposed to AraC from 4 days onwards. There was minimal degeneration in the 4-7 day treated explants. Electron microscopic examination revealed the presence of atypical inclusions in non-neuronal cells of 4-8 day treated explants, suggesting that the cytotoxic effect of AraC may be due to a disturbance in lipid and/or ganglioside metabolism. Quantitative electron microscopic analysis of the outgrowth zone at 18 DIV revealed a significant increase in the summated area of neuronal tissue (from 7 to 18 k~m2/100 Nm 2) and a decline in the summated area of non-neuronal cells (from 83 to 61 #m2/100/,tin 2) for explants treated with AraC from 4 to 7 DIV compared to control. Diminishing the potential of non-neuronal cells to act as a barrier by controlling their proliferation may, therefore, be of importance in enhancing the regenerative response of central neurons.

Journal of neurophysiology, 1977

1. Reliable methods for establishing fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) cells in long term (greater than 1 mo) dissociated cell cultures are described. These cells have been studied by morphologic and intracellular electrophysiologic techniques. 2. Cells studied electrophysiologically can be relocated after preparation for electron microscopy and examined in thin sections. The electron microscope shows that the surface membranes of these cells were directly accessible to the culture medium. The surfaces of SC cells were studded with synaptic boutons, whereas the DRG cell surfaces generally had none. 3. Current-voltage relationships and linear electrotonic properties of the neurons are described. Delayed and anomalous rectification were seen in both cell types. The length of SC cell dendrites was about one characteristic electrotonic length, while little or no contribution of the relatively sparse DRG cell processes was seen in the transient responses of the ...

Biochimica et Biophysica Acta (BBA) - Enzymology, 1973

I. The development of the lysosomal hydrolase N-acetyl-fi-D-glucosaminidase was examined in bulk-isolated nerve cell bodies of rat cerebral cortex. Several properties of the enzyme and of two of its molecular components were compared, among them their ease of solubilization, their relative abundance, their heat lability and their behavior on density gradients of sucrose. 2. Repeated freezing-thawing of neuronal particulates in o.2o M sucroseo.I M KC1 followed by centrifugation at moderate rather than high speeds resulted in the solubilization of upward of 5o% of the glucosaminidase, irrespective of neuronal age. 3. Although the solubilization of N-acetyl-fl-D-glucosaminidase was dependent on the concentration (mg of protein/ml) of the suspension subjected to freezingthawing, it was always more effective with the enzyme from the 8-and IS-day-old than with that from the 3-and 5-day-old neurons. 4. Centrifugation of the solubilized neuronal N-acetyl-fl-D-glucosaminidase on linear density gradients of sucrose resulted, at all ages, in the complete resolution of two activity components a heavy and a light one, H and L respectively. As routinely isolated, the ratio of component L to component H in the neurons of the cerebral cortex was always >I.O while, in the cerebellar neurons, values < I.O were also noted. 5-The qualitative and quantitative gradient profiles of N-acetyl-fl-D-glucosaminidase activity were highly sensitive to pH. Thus, lowering of the pH of frozen thawed supernatants to 5.4 and below prior to density gradient centrifugation resulted in the virtual disappearance of component H, while adjustment of the pH to 5.6 resulted in L/H ratios <I.o and in the appearance of additional, smaller peaks of N-acetyl-fl-D-glucosaminidase activity. 6. About 5o% of the activity of component L was lost, irrespective of age, by heating for 2o min at 5o °C and at a pH of 4.1. The heat sensitivity of component H was no greater, except at 5 days, when about 90% of its activity was lost under the same conditions.

Proceedings of the National Academy of Sciences of the United States of America, 1969

Mouse tumor C1300 has been established in tissue culture. The cells have a round cell morphology in both the subcutaneous tumor and in suspension culture. However, when given a surface on which to attach, they send out processes up to 3 mm in length and assume the morphology of mature neurons. The attached cells are stained by the Bodian silver procedure for neurons, whereas the cells grown in suspension are not. Electron microscopy reveals that the attached cells contain neurofilaments, neurotubules, and densecore vesicles indicative of nerve fibers. Both free-floating and attached cells have tyrosine hydroxylase activity characteristic of sympathetic nervous tissue. Apparently cell attachment can induce morphological differentiation from an anaplastic round cell to a cell which has many properties of a mature neuron.

Journal of Neuroscience Research, 1989

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is Iayered onto a nylon mesh with a pore size of 10 pm. Most of the neurons, the diameter of which ranged from 17 pm to > 100 pm, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of < 10 pm after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 6040% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia. As NGF has no survival-promoting effect on these purified adult neurons, the trophic effect of CMs is likely to be mediated by non-NGF molecule(s).

The Journal of Cell Biology, 1976

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasma...

International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 1985

Mouse neural tube development in vitro was examined following the isolation and culturing of specific regions of the neural tube. Developmental characteristics of neuron formation and differentiation were assessed both quantitatively and qualitatively. A 1 mm length of embryonic day 10 mouse neural tube was cut into 32 microfragments of equal size and cultured on collagen coated cover-slips. Neuronal cells were observed to emerge after 3 days in culture and to migrate away from the fragment upon an immature astroglial precursor cell layer that had begun to form within the first 24 h of culturing. The extent of neuronal migration, the density and total number of neurons per outgrowth zone, and the size distribution of neurons was quantitated after 21 days in culture. Three distinct patterns of neuronal outgrowth (Types II, III and IV) could be observed with a fourth pattern (Type I) best described as being neuron free. Neuron free outgrowth zones (Type 1) were comprized totally of gl...

The Journal of neuroscience : the official journal of the Society for Neuroscience, 1988

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Developmental Biology, 1982

Embryonic chicken sensory cells from dorsal root ganglia and a clonal line of pheochromocytoma cells (PC-12) extended neuronal-like processes within 24 hr of seeding on a naturally produced, basement membrane-like extracellular matrix (ECM) in the absence of nerve growth factor (NGF). Plating on ECM also induced a rapid cell attachment and flattening of these cells and supported the survival of embryonic sensory cells in primary cultures, Unlike the effect of NGF on PC-12 cells, the ECM-induced morphological differentiation was transient and led to disintegration and degeneration of processes bearing PC-12 cells. The ECM-induced morphological differentiation was not inhibited by anti-NGF antibodies, and the cells retained their ability to bind and internalize NGF in a manner similar to that observed on plastic. PC-12 cell attachment and flattening occurred on dishes coated with collagen type IV in a way similar to that observed on ECM, but precoating the dishes with tibronectin had no effect. Extension of cell processes was not induced by either substrate. Morphological differentiation but not the induction of cell adhesion and flattening was inhibited by either prefixation with glutaraldehyde, oxidation with periodate, or preexposure to concanavalin A of the ECM, suggesting that the ECM and in particular its sugar moieties play an active role in the induction of neurite outgrowth. It is suggested that close contact with the ECM provides chemical or mechanical cues that permit contactmediated elongation and directed growth of both embryonic and regenerating nerve fibers.

Annals of the New York Academy of Sciences, 1986