Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons.

To investigate the biological role of basic fibroblast growth factor (bFGF) for the development of the spinal cord we studied the in vitro and in vivo effects of this protein on survival and choline acetyltransferase (ChAT)-activity of embryonic chick and rat spinal cord neurons. In vitro, bFGF (ED50 1-2.8 ng/ml) supported the survival of embryonic neurons from the ventral part of the rat spinal cord (ventral spinal cord, vsc), including motoneurons. Addition of bFGF (100 ng/ml) increased the ChAT-activity in embryonic chick vsc cultures to 150% as compared to untreated cultures (100%). The effect of bFGF was dose-dependent. In vivo-application of bFGF resulted in a similar increase of ChAT-activity in chick spinal cord. Since bFGF stimulates the ChAT-activity of spinal cord neurons in vivo and in vitro we therefore conclude that this protein may have a physiological function for the transmitter development of cholinergic spinal cord neurons.     

CNTF rescues motoneurons from ontogenetic cell death in-vivo, but not in-vitro

We studied the effect of CNTF (ciliary neurotrophic factor, human recombinant and chick) on the survival of motoneurons in the embryonic chick lumbar spinal cord during the period of ontogenetic cell death. Daily applications of 5 micrograms CNTF to the chorionic-allantoic membrane from embryonic day 6 (E6) to E9 maintained approximately 15,500 motoneurons as opposed to 13,200 in controls. In contrast, CNTF failed to promote the survival of cells in spinal cord cultures enriched for motoneurons. These results suggest that CNTF may regulate motoneuron survival in-vivo, but its mode of action remains to be elucidated.     

Inhibition of conditioned media-mediated neuritogenesis of sensory ganglia by monoclonal antibodies to GM1 ganglioside

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 degrees C for 5 h in NM supplemented with 150 micrograms/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons

Acetylcholine metabolism has been studied in sister cultures of E13 rat spinal cord cells cultured for 1 to 3 weeks with or without conditioned medium (CM) from rat skeletal muscle cells. Spinal cord cells grown with CM synthesized and accumulated 3 to 4 times more [3H]ACh from [3H]choline than cultures grown without CM. This effect of CM was accompanied by a comparable increase in CAT activity and could not be mimicked by increasing the density of the spinal cord cultures. A 2- to 3-fold increase in AChE activity was also observed in 2- to 3-week-old CM cultures, whereas the activity of lactate dehydrogenase was identical in cultures grown with and without CM. We have compared the effects of CMs from various non-neuronal cell cultures on [3H]ACh synthesis and storage by spinal cord cultures and by sympathetic neuron cultures. CM by skeletal muscle greater than skin fibroblasts greater than rat heart muscle greater than C6 glioma cells were the most active on both types of neuron cultures, whereas CM from rat brain, L6 myoblasts, mouse 3T3, and PYT21 fibroblasts was inactive on spinal cord cultures and only weakly active on sympathetic neurons. Serum-free CM from skeletal muscle was inactive on both types of neuron cultures. The CM factor active on spinal cord cultures has been purified several thousand-fold by using a four-step fractionation scheme which has previously led to a partial purification of the CM factor involved in the regulation of CAT, AChE, and catecholamine-synthesizing enzymes in sympathetic neuron cultures ( Swerts , J. P., A. Le Van Thai, A. Vigny , and M. J. Weber (1983) Dev. Biol. 100: 1-11). Moreover, a comparison of dose-response curves established with this purified material showed that it exerted its effects on spinal cord and on sympathetic neuron cultures in the same range of concentration. Thus, these results suggest that the same macromolecule is involved in the regulation of neurotransmitter phenotype in both types of cultures despite their different embryological origins.     

The influence of skeletal muscle on the electrical excitability of dorsal root ganglion neurons in culture

Dorsal root ganglion (DRG) neurons from embryonic mice grown in coculture with dissociated skeletal muscle or in skeletal muscle conditioned medium (CM) showed an increased incidence of repetitive firing of action potentials when injected with sustained (60-100 msec) depolarizing current. This is in contrast to DRG neurons grown in monoculture and normal medium, which exhibit such behavior far less frequently. The first action potential showed less sensitivity to block with TTX and more sensitivity to Ca2+ channel blockers than the subsequent action potentials. The increased incidence of repetitive firing occurred when CM was added after as few as 2 or as many as 22 d in culture and with as little as 1-7 hr exposure to CM. This effect of CM cannot be mimicked by NGF or by coculture with cells from embryonic spinal cord (Peacock et al., 1973), can be eliminated by heating the CM at 56 degrees C for 30 min, and partially reversed following short exposure to CM. These results indicate that skeletal muscle releases some heat-labile factor(s) that can cause repetitive firing and, in addition, significant decrease in input resistance in the CM-treated neurons and a depression of the anomalous rectification, neither of which could account for the increase in repetitive firing.     

Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons

We have recently reported the existence within chick embryo heart cell conditioned medium (HCM) of two distinct and independently assayable factors. One agent, ciliary neuronotrophic factor (CNTF), supports the in vitro survival of 8-day chick embryo ciliary ganglionic (CG) neurons. The other factor, polyornithine-attachable neurite promoting factor (PNPF) is required for extensive neuritic growth from these same CNTF-supported CG neurons.In the present study we have examined the occurrence of PNPF activity within nearly 100 different conditioned media using our previously described chick CG bioassay system. From this screening we conclude that: (1) PNPF production is a rather widespread property of cultured neural as well as non-neural cells; and (2) the chick bioassay is sensitive to PNPF activity from all the species examined, including mouse, rat, human and chick cells.We next examined the effects of 3 representative PNPF-containing conditioned media (from chick heart, mouse Schwann and rat Schwannoma) on neurite production from 3 other peripheral ganglionic neuronal cultures (8-day chick dorsal root, 11-day chick sympathetic, and neonatal mouse dorsal root ganglia) as well as 4 central neuronal cultures (8-day chick embryo telencephalon, optic lobe and spinal cord and neonatal mouse cerebellum). The results of these studies indicate: (1) that the peripheral neurons exhibit a dramatic increase in neurite production in response to PNPF which can be easily recognized both qualitatively and quantitatively; whereas (2) the CNS neurons showed essentially no PNPF-induced increase in neurite production. The sole exception to the latter was the appearance within the chick spinal cord cultures of a neuronal population which extended very long neurites in response to PNPF.     

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.     

Biological properties of crude growth inhibiting factor produced by embryonic spinal cord cells in culture

Cultured chick spinal cord cells produced a soluble factor or factors which inhibited growth and development of muscle cells in culture. The activity of cord inhibitory factor depended on the number of cord cells inoculated into culture, and the duration of their incubation in culture. Nonreplicating frozen and thawed cells produced little or no inhibitory factor. Cord inhibitory factor was produced by chick cord cells from embryos tested at 11 to 21 days of age. While “stale” media from thigh, heart, and skin cell cultures also were slightly inhibitory, their activity was several-fold less potent than that of cord cells. Media collected from liver cell cultures stimulated growth and protein synthesis. Crude cord inhibitory factor had little or no effect on target muscle cells (plated at 1 × 10⁶ cells per dish) after the first 2 days of culture. Cells began in culture at lower density, however (1 × 10⁵ cells per dish), retained their susceptibility for at least 4 days in culture while continuing active DNA synthesis. This suggests that cord inhibitory factor acted on cells while they were replicating. Although inhibited cells did not incorporate [¹⁴C]lysine into protein normally, they retained the ability to take up the amino acid into their cytoplasm. Inhibitory activity of crude cord factor was also demonstrable against cultures of heart and skin cells. Cord cell cultures, however, were relatively resistant. It is at present not known whether the inhibitory factor is produced by neuronal or glial cells.     

Androgen action in fetal mouse spinal cord cultures: metabolic and morphologic aspects

Morphologic and metabolic aspects of androgen effects on the developing spinal cord were studied in organotypic cultures of the E13 (embryonic day 13) fetal mouse lumbosacral spinal cord, maintained as either hemisected, homologous explant pairs co-cultured with bulbocavernosus muscle (morphologic studies), or as whole cross-sectional segments without muscle in which the nutrient medium was supplemented with muscle extract (metabolic studies). Metabolic studies demonstrated the total absence of aromatase activity. 5 alpha-Reductase activity, on the other hand, increased differentially in a segment-dependent manner in spinal cord explants from 0 to 35 days in culture, suggesting regional differences in the utilization of testosterone and its 5 alpha-reduced metabolites. In all studies, spinal cord explants showed androgen-dependent increases in neurite outgrowth, although this was most pronounced in spinal cord-muscle co-cultures. These results indicate that androgens per se affect very early development throughout the entire lumbosacral spinal cord, and that this influence is not restricted to those segments reported to be sexually dimorphic in the adult.     

Isolation of embryonic chick motoneurons and their survival in vitro

This is the first of a series of 4 papers in which we describe the regulation of excitatory amino acid receptors on embryonic chick motoneurons dissociated from the lateral motor column and maintained in cell culture. Techniques are described for labeling embryonic chick motoneurons in vivo with Lucifer Yellow or fluorescein isothiocyanate conjugates of wheat germ agglutinin (Fl-WGA). We estimate that 65-95% of the motoneurons in the lateral motor column survive tissue dissociation and settle on an appropriate culture surface. The number of fluorescent motoneurons observed in heterogeneous spinal cord cell cultures decreases with a half-life of 2 d. The decline is due to fading of the fluorescent tracer rather than to loss of cells. Techniques are also described for separating motoneurons from other spinal cord cells with a fluorescence-activated cell sorter. Approximately 24% of the motoneurons in the lateral motor column can be isolated, and motoneurons comprise more than 90% of the population in cultures seeded with sorted cells. The survival of sorted and unsorted motoneurons in vitro is enhanced in the presence of skeletal myotubes or muscle conditioned medium, but the survival of non-motoneurons is not influenced by muscle. Electrophysiologic properties of sorted and unsorted motoneurons determined with patch-clamp techniques are similar. Both differ from mature motoneurons in their lower resting membrane potential (-50 mV), larger input resistance (450 M omega), and longer time constant (39 msec). Also they do not exhibit anomalous rectification or a calcium-activated potassium after hyperpolarization. Motoneurons grown in the absence of interneurons differ from motoneurons in heterogeneous spinal cord cell cultures in that their neurites (dendrites) are shorter and they branch less often.     

Gangliosides of the mouse spinal cord: a comparison in in vivo and in vitro tissues

Ganglioside profiles in spinal cord from 13-day mouse fetuses, 21-day postnatal and adult mice were compared with those harvested from organotypic cross-sections of fetal mouse spinal cord grown for 28 days in vitro in a serum-free medium. All the major species of gangliosides reported for brain were present both in the in vivo tissue and cultured spinal cord, though not necessarily at each developmental stage examined. Fresh tissues showed increases and decreases in various gangliosides as have been reported for higher brain centers at similar stages of development in mammals and birds. However, qualitative and quantitative differences exist between fresh spinal cord and cultured cord explants as well as between galactose-grown and galactose-free cultures. Spinal cord explants grown in the presence of galactose showed measurable amounts of GM2 and GM3 which were not detected in the control-defined medium-grown cultures. The differences between the two culture groups may be related to interneuronal connectivity patterns.     

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Analysis of the change in number of serotonergic neurons in the chick spinal cord during embryonic development

The existence of serotonin (5-HT)-containing neurons in the spinal cord of the chick embryo was examined by anti-5-HT immunocytochemistry. The first immunoreactive cells were observed in embryos at 7 days of incubation (E7) and were initially located within the floor plate of the early spinal cord. By E9, immunostained cells occurred throughout the length of the spinal cord and were frequently encountered in most transverse sections of the cord. When examined at later embryonic ages of E12, 17 and at hatching (E21 or 22), the 5-HT cells became progressively more difficult to find with the advancing age of the embryos. To determine if this population of spinal cord 5-HT neurons actually diminished during development, a detailed quantitative analysis was undertaken to estimate the number of 5-HT cells in the cord of chick embryos at different ages. The results of this investigation demonstrated that the size of the 5-HT neuronal population rose rapidly from E7 and plateaued (at approximately 3500 neurons) between E9 and E12. As anticipated, the number of 5-HT cells at E17 decreased at all cord levels. Surprisingly, however, the number of spinal cord 5-HT neurons at hatching increased (depending on the cord level) either back to, or above, the counts estimated for the earlier ages of E9 and E12. Therefore, cells expressing the 5-HT phenotype in the spinal cord of the chick embryo persist throughout the period of embryonic development, rather than appear transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of spinal cord growth inhibitor by nonneuronal cells

Separation of chick embryo spinal cord cells into cell cultures of glial-enriched and neuronal-enriched elements by the method of selective adhesiveness indicated a greater production of growth inhibitor by the glial-enriched cultures. Glial-enriched cultures produced 177.2% more inhibitor per microgram cell protein than did unseparated spinal cord cell cultures, whereas neuronal-enriched cultures produced less. The glial-enriched cultures contained 28% of the choline acetyltransferase of neuronal-enriched cultures, few or no discernible neurons, and no cells binding dopamine or containing catecholamines by cytofluorescence. The predominant cell of the glial-enriched cultures was large, phase-light with ruffled borders, with parallel streak-like processes, and fibrillar cytoplasm. Cultures produced by serial passage of unseparated spinal cord cells also were found to be depleted in neurons and dopamine-binding cells, as well as in choline acetyltransferase activity, to 19% of control values. These serially passaged cultures, however, retained the ability to produce inhibitor as well as did primary cultures. Serially passaged cultures of spinal cord cells were composed of cells similar to those predominant in glial-enriched cultures. Nerve growth factor had no effect on inhibitor production. We suggest that the growth inhibitor of spinal cord cultures was not produced by neuronal cells but may be of glial origin.     

Notochord and floor plate stimulate oligodendrocyte differentiation in cultures of the chick dorsal neural tube

The regionalization of oligodendrocyte potentialities and the cellular interactions leading to the expression of the oligodendrocyte phenotype have been analyzed in the embryonic chick spinal cord. Dorsal and ventral regions of the spinal cord of 4-day-old embryos (E4) were cultivated separately. Oligodendrocyte differentiation was monitored at various times after explantation, using specific oligodendrocyte markers. After 2 weeks, several hundreds of differentiated oligodendrocytes were invariably observed in ventral cultures whereas significant numbers of oligodendrocytes failed to develop in dorsal spinal cord cultures. However, the E7 dorsal spinal cord was found to produce large numbers of oligodendrocytes, indicating that the ventral restriction of oligodendrocyte potentialities is transient. To test whether ventrally derived signals might influence oligodendrocyte differentiation, E4 dorsal spinal cord microexplants were cocultivated with notochord segments or with floor plate tissue. Numerous oligodendrocytes were found in dorsal explants associated with either tissue, notochord or floor plate, but not in dorsal explants cultivated alone, indicating that cells competent to be induced along the oligodendrocyte phenotype exist in the dorsal spinal cord. These results show that oligodendrocyte differentiation potentialities are initially restricted to the ventral spinal cord and suggest that ventrally derived signals from notochord and floor plate influence oligodendrocyte differentiation in the embryonic spinal cord. © 1995 Wiley-Liss, Inc.     

Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors

Besides their capacity to give rise to neurons and/or glia, neural stem cells (NSCs) appear to inherently secrete neurotrophic factors beneficial to injured neurons. To test this potential, we have implanted NSCs onto or adjacent to spinal cord cultures. When NSCs were placed adjacent to the spinal cord sections, motor neuron axons grew toward the NSCs. Furthermore, conditioned medium from NSCs cultures was also able to induce similar axonal outgrowth, suggesting that these NSCs secrete soluble factors that have tropic and/or trophic properties. ELISA revealed that the NSCs secrete glial cell-line-derived factor (GDNF) and nerve growth factor (NGF). Interestingly, preincubation of the conditioned medium with GDNF-blocking antibodies abolished axonal outgrowth. We also showed that NSCs can protect spinal cord cultures from experimentally induced excitotoxic damage. The neuroprotective potential of NSCs was further confirmed in vivo by their ability to protect against motor neuron cell death.     

Ganglioside GM1 overcomes serum inhibition of neuritic outgrowth

Considerable variations in both neuritic and non-neuronal cell outgrowths of ganglionic expiant cultures are imposed when different substrata and medium supplements are used. Therefore, a neuritic response to exogenous agents such as gangliosides may be better, or exclusively revealed under selected combinations of medium and substratum. We have surveyed a number of different ganglia for their ability to display a neuritic response to ganglioside GM1 and the culture conditions under which such a display may take place. GM1 can recognizably promote neurite extension from embryonic day 8 chick dorsal root ganglia, day 11 sympathetic ganglia, and day 8 ciliary ganglia. Demonstration of such effects, however, requires an appropriate balance between promoting and inhibiting influences by other extrinsic influences (culture substrata, neuronotrophic factors, serum inhibitors) thereby allowing for improvement by the ganglioside. Different ganglia require different combinations of those influences. Ciliary ganglia, for example, will show a GM1 response on a polyornithine substratum in medium supplemented with the chick eye Ciliary Neuronotrophic Factor and 1% fetal calf serum, but not with 10% serum or no serum. The effective concentration range for GM1 is in accord with previously reported serum binding data. These, and other data are consistent with an action of GM1 in executing a neurite program, rather than an imposition of the program itself.     

Endogenous and exogenous factors support neuronal survival and choline acetyltransferase activity in embryonic spinal cord cultures

Dissociated 4-day (stage 23) chick embryo lumbar cord cells were cultured at low or high cell densities for 1 or 5 days in the presence or absence of added spinal neuronotrophic factor (supplied as RN22 Schwannoma conditioned medium, RCM). In low density, 1-day cultures neuronal survival was dependent on added RCM whereas by 5 days no neurons survived, even in the presence of RCM. In high density 1-day cultures a substantial neuronal population could survive even without added RCM and a large proportion of this neuronal population would survive for 5 days. When conditioned media from high density lumbar cord cultures was supplied to low density unsupplemented cultures, a similar level of 5-day neuronal survival resulted. However, no neurons survived in RCM-supplemented 5-day high density cultures, indicating the presence in RCM of a material toxic for the neurons. Both the RCM and the high density lumbar culture-conditioned medium supported considerable choline acetyltransferase activity indicating the presence within these cultures of motoneurons.     

Leukaemia Inhibitory Factor or Related Factors Promote the Differentiation of Neuronal and Astrocytic Precursors within the Developing Murine Spinal Cord

Previously we have shown that leukaemia inhibitory factor (LIF) potentiates the development of murine spinal cord neurons in vitro , suggesting that it, or related factors, may play an important regulatory role in neuronal development. We have further investigated this role and show here that the generation of neurons in cultures of embryonic day 10 spinal cord cells is inhibited by antibodies to the β subunit of the LIF receptor. Since there are more undifferentiated precursors in antibody-treated cultures than in control and LIF-treated cultures, it is concluded that the primary action of LIF, or related molecules, is to promote neuronal differentiation, not precursor survival. In addition, the failure of LIF to support neuronal survival in the period immediately following differentiation suggests that the increased numbers of neurons generated with LIF are not attributable to its neurotrophic action. By selecting neuronal precursors on the basis of their inability to express class I major histocompatibility complex molecules, it was shown that LIF acted directly upon these cells and not via an intermediary cell. LIF also appears to be involved in regulating the differentiation of astrocytes, since it increases the number of glial fibrillary protein (GFAP)-positive cells present in the cultures and since the spontaneous production of GFAP-positive cells is blocked by antibodies to the LIF β receptor. These findings suggest that LIF or related factors promote the differentiation of neural precursors in the spinal cord, but that they are not involved in preferentially promoting precursors down a specific differentiation pathway.