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.     

Interferon stimulates the expression of cholinergic properties in human spinal cord neurons in culture

A preparation of dissociated monolayer cultures from embryonic human spinal cord has been developed and characterized (Kato, Touzeau, Bertrand, Bader, 1985) as a model system for the study of amyotrophic lateral sclerosis (Touzeau and Kato, 1986). The cultures contain cholinergic and GABAergic neurons, astrocytes and fibroblasts. We have recently found that gamma-interferon (IFN) can increase the choline acetyltransferase (CAT) activity without altering the level of glutamic acid decarboxylase (GAD) or the neuronal survival; an antibody to IFN can prevent these effects. Gamma-IFN appears to mediate these effects via the non-neuronal cells since in the absence of non-neuronal cells, gamma-IFN has no effect on the cholinergic properties. The non-neuronal cells alone have no CAT or GAD activity. Astrocytes may be responsible for these changes since gamma-IFN increases the development of GFAP immunoreactivity in cultures of 6-7 week old spinal cord cells and it causes no visible change in the Thy-1 immunoreactivity of the fibroblasts. Thus we propose that IFN acts on non-neuronal cells, possibly the astrocytes, which in turn stimulate neuronal cholinergic traits either by means of a diffusible factor or via cell-cell contact. These studies could be relevant in understanding the effects of the immune system on the nervous system and also in the search for new drugs which act specifically on cholinergic neurons.     

Potent pro-inflammatory actions of leukemia inhibitory factor in the spinal cord of the adult mouse

Injury in the peripheral or central nervous systems causes a significant rise in the levels of the pleiotropic cytokine leukemia inhibitory factor (LIF). This increase influences cell survival, reactive gliosis and inflammatory responses. Since prior work has focused primarily on peripheral nerve and brain, little is known about the role of LIF in the spinal cord injury response. We address this issue by examining the effects of injury in the LIF knockout (KO) mouse, as well as using an adenoviral vector to over-express LIF in the spinal cord of adult mice. We find that LIF over-expression results in a dramatic rise in cell proliferation, primarily in microglia/macrophages. Astrocytes are not stimulated to proliferate but are activated by the elevated LIF. LIF over-expression also causes the development of severe hindlimb motor dysfunction, an effect mediated by the enhanced activation of microglia/macrophages, as inhibiting microglial activation with minocycline attenuates these motor deficits. Conversely, proliferation is significantly diminished and the microglial/macrophage response to spinal cord injury is much less in the LIF KO compared to wild type (WT). Thus, LIF is a potent pro-inflammatory factor in the adult spinal cord and represents a potential target for the manipulation of inflammatory reactions after spinal cord injury.     

Schwann cell-conditioned medium supports neurite outgrowth and survival of spinal cord neurons in culture

The effect of Schwann cell-conditioned medium (SCM) on the development in vitro of spinal cord neurons was studied. Spinal cord neurons from 18-day-old rat embryos were cultured in serum-free conditioned medium obtained from confluent rat Schwann cells. In cultures fed SCM, the cells developed typical neuronal morphology and were identified by indirect immunofluorescence using a monoclonal antibody to neurofilament protein. SCM stimulated neurite outgrowth and supported survival of spinal cord neurons. Preliminary characterization suggests that the neurotrophic factor in SCM appears to be a protein with a molecular weight greater than 8000 daltons.     

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.     

Retinoic acid responsive gene product,midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley-Liss, Inc.     

Sonic hedgehog promotes neuronal differentiation of murine spinal cord precursors and collaborates with neurotrophin 3 to induce Islet-1.

Sonic hedgehog (Shh) is strongly implicated in the development of ventral structures in the nervous system. Addition of Sonic hedgehog protein to chick spinal cord explants induces floor plate and motoneuron development. Whether Shh acts directly to induce these cell types or whether their induction is mediated by additional factors is unknown. To further investigate the role of Shh in spinal neuron development, we have used low-density cultures of murine spinal cord precursor cells. Shh stimulated neuronal differentiation; however, it did not increase the proportion of neurons expressing the first postmitotic motoneuron marker Islet-1. Moreover, Shh did induce Islet-1 expression in neural tube explants, suggesting that it acts in combination with neural tube factors to induce motoneurons. Another factor implicated in motoneuron development is neurotrophin 3 (NT3), and when assayed in isolated precursor cultures, it had no effect on Islet-1 expression. However, the combination of N-terminal Shh and NT3 induced Islet-1 expression in the majority of neurons in low-density cultures of caudal intermediate neural plate. Furthermore, in explant cultures, Shh-mediated Islet-1 expression was blocked by an anti-NT3 antibody. Previous studies have shown expression of NT3 in the region of motoneuron differentiation and that spinal fusimotor neurons are lost in NT3 knock-out animals. Taken together, these findings suggest that Shh can act directly on spinal cord precursors to promote neuronal differentiation, but induction of Islet-1 expression is regulated by factors additional to Shh, including NT3.     

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding and retrograde transport of leukemia inhibitory factor by the sensory nervous system.

Leukemia inhibitory factor (LIF), a peptide growth factor with multiple activities, has recently been shown to support the generation and survival of sensory neurons in cultures of mouse neural crest and dorsal root ganglia (DRG). We have conducted binding experiments with 125I-LIF on cultures of DRG to determine the receptor distribution for LIF on these cells and found that at least 60% of the sensory neurons in the cultures bound 125I-LIF, all of which could be eliminated by the addition of unlabeled LIF. The other cells in the culture, which morphologically appeared to be Schwann cells, did not bind appreciable quantities of 125I-LIF. In order to investigate whether LIF is retrogradely transported to sensory neurons in vivo, 125I-LIF was injected into the footpads and gastrocnemius muscles of newborn and adult mice, following sciatic nerve ligation. Radioactivity accumulated in the distal portion of the sciatic nerve, indicating retrograde transport of LIF. Subsequent experiments on mice with unligated sciatic nerves showed that 125I-LIF is specifically transported into the sensory neurons of the DRG. There was no apparent transport of 125I-LIF into motor neurons in the spinal cord. These experiments demonstrate that LIF can specifically bind to and be transported by sensory neurons and further support the idea that LIF acts as a target-derived neurotrophic factor, analogous to NGF.     

Expression of insulin‐like growth factor‐II and leukemia inhibitory factor antibody immunostaining on the ionized calcium‐binding adaptor molecule 1‐positive microglias in the spinal cord of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease involving the upper and lower motor neuron systems. Activated microglia are reported to enhance motor neuron death by secreting neurotoxic cytokines in SOD1‐transgenic mice. Recent studies have provided evidence that chronic stimulation leads microglia to acquire an anti‐inflammatory phenotype, characterized by activated morphology and induction of neuroprotective and immunoregulatory molecules. However, little information is available on the protective functions of microglia in the ALS spinal cord. To investigate the roles of microglia in ALS, we examined the appearance of ionized calcium‐binding adaptor molecule 1‐positive (Iba1‐positive) microglia as correlated to the disease duration and immunohistochemical expression of neurogrowth factors in the ALS spinal cord. In this study, the number of Iba1‐positive rod‐like microglia significantly increased in the ALS spinal cord compared to controls. The number of ramified microglia was positively correlated with the number of normal‐looking neurons and clinical duration of ALS patients; however, the number of rod‐like microglia was not correlated with that of abnormal neurons, nor with the clinical duration of the disease. Some rod‐like microglia were positive for anti‐insulin‐like growth factor‐II (IGF II) and anti‐leukemia inhibitory factor (LIF) immunostaining. Motor neurons in the ALS spinal cords also showed immunoreactivity for IGF‐II, LIF and the receptors of IGF‐II and LIF. Taken together, these findings suggest that at least some microglia might have a protective effect on motor neurons in the ALS spinal cord. Neuroprotective and/or neurotoxic effects of microglia on motor neurons should be further studied.     

Differential modulation of the cholinergic activity of rat CNS neurons in culture.

Treatment of septal cultures prepared from 17-day-old embryos with two different antimitotic agents, cytosine arabinoside (ara C) and 5'-fluoro-2'-deoxyuridine (FUdR), caused a 2-fold increase in the level of choline acetyltransferase (CAT) activity and no change in the glutamic acid decarboxylase (GAD) activity. In these cultures, there was also a large decrease in the number of astrocytes as determined by immunofluorescence for glial fibrillary acidic protein (GFAP). Furthermore, when epidermal growth factor (EGF) was added to the septal cultures to increase the astrocyte population, the CAT activity decreased. Therefore, it would appear that the astrocytes are responsible for producing this down-regulation on cholinergic neurons. In order to determine whether all CNS cholinergic neurons can be inhibited in this manner, cultures were prepared from two other CNS regions that contain a high percentage of cholinergic neurons, i.e. the striatum and the ventral spinal cord. When these cultures were treated with the antimitotic agents, there was little modification of the CAT or GAD activities. These results suggest that the astrocytic microenvironment of the septal neurons exerts an inhibitory effect on the CAT activity either via a soluble factor or via cell-cell contact. Such studies are an important demonstration that non-neuronal cells may alter cholinergic properties during CNS development.     

Human spinal cord neurons in dissociated monolayer cultures: morphological, biochemical, and electrophysiological properties

The preparation of dissociated monolayer cultures from embryonic human spinal cord is described. Optimal survival was achieved with embryonic tissue between the eighth and ninth week. The neurons survive for as long as 7 weeks in culture and they grow in a standard tissue culture medium which contains 13% decomplemented human serum. The neurons have been identified by indirect immunofluorescence techniques using antibodies to tetanus toxin and neurofilament protein. Our biochemical studies demonstrate the presence of cholinergic and GABAergic neurons. Cholinergic neurons develop in culture and are more numerous in the cultures prepared from the anterior part of the spinal cord as compared to those from the posterior part. Therefore, it is possible that a large part of the cholinergic neurons derive from the motoneuron pool. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin (TTX). Voltage clamp experiments combined with the use of pharmacological blocking agents revealed the presence of several voltage- and time-dependent currents: a sodium current sensitive to TTX, a potassium current made up of two components, sensitive to tetraethylammonium and 4-aminopyridine, and a calcium current sensitive to cobalt. From a biochemical and electrophysiological point of view the properties of human spinal cord neurons in culture closely resemble the properties of spinal cord neurons from other species.     

Histochemical and electrophysiological evidence for estrogen receptors on cultured astrocytes: colocalization with cholinergic receptors

By means of autoradiographic and immunohistochemical methods it was demonstrated that astrocytes in explant and primary cultures of rat neocortex, hippocampus, preoptic area and spinal cord express estrogen alpha- and beta-receptors. Immunoreactivity was mainly distributed over the soma, the nuclei being more intensely stained. Combined autoradiographic and immunohistochemical studies as well as double-immunostaining revealed a colocalization of estrogen alpha- and beta-receptors on many astrocytes. There was also a coexistence of estrogen receptors and cholinergic muscarinic and nicotinic sites. Electrophysiological investigations have shown that 17beta-estradiol induced hyperpolarizations on the majority of astrocytes in explant cultures of hippocampus and spinal cord, providing evidence for the existence of functional estrogen receptors on these cells. Furthermore, on the same astrocytes, 17beta-estradiol, muscarine and nicotine caused hyperpolarizations, suggesting a coexistence of receptors for estrogen and the cholinergic agonists on glial cells. The presence of glial estrogen receptors and their colocalization with cholinergic receptors is discussed with respect to the effects of these neurotransmitters/neuromodulators in development and maturation of the central nervous system, as well as to neurodegenerative events such as Alzheimer's disease.     

Preganglionic neurons from the Edinger-Westphal nucleus: growth and histochemical characterization in cell culture

The Edinger-Westphal (EW) nucleus, also known as the accessory oculomotor nucleus in the chick, provides the cholinergic preganglionic input to the parasympathetic ciliary ganglion. In addition to acetylcholine, many EW neurons have been shown to contain enkephalin-like and/or substance P-like immunoreactivity. Establishment of EW neurons in culture would make possible study of their interactions with ciliary ganglion neurons in vitro and in addition would provide a valuable system for studying cholinergic/peptidergic neurons of the vertebrate central nervous system. We describe here dissociated cell cultures established from midbrain tissue containing the EW nucleus. In these cultures, 86% of the cells with neuronal morphology were positive for intracellular acetylcholinesterase activity, 54% were positive for enkephalin-like immunoreactivity, and 4% were positive for substance P-like immunoreactivity. The proportions of neurons that scored as labeled were even higher if the number of positive cells was compared to the number of cells in sister cultures immunoreactive for the large neurofilament protein polypeptide. When the cultures were stained simultaneously for acetylcholinesterase activity and enkephalin-like immunoreactivity, 34% of the cells with neuronal morphology were positive for both. In cultures derived from adjacent tissue regions very few cells expressed both activities. These results suggest that the cells expressing both acetylcholinesterase activity and enkephalin-like immunoreactivity in culture are EW neurons. The putative EW neurons survive for weeks in vitro in the absence of their normal target, the ciliary ganglion.     

A culture model for neurite regeneration of human spinal cord neurons

Effective therapeutic interventions for injuries of the central nervous system such as spinal cord injury are still unavailable, having a great impact on the quality of life of victims and their families, as well as high costs in medical care. Animal models of spinal cord injury are costly, time-consuming and labor-intensive, making them unsuitable for screening large numbers of experimental conditions. Thus, culture models that recapitulate key aspects of neuronal changes in central nervous system injuries are needed to gain further understanding of the pathological and regenerative mechanisms involved, as well as to accelerate the screening of potential therapeutic agents. In this study we differentiated adherent cultures of dissociated human fetal spinal cord neural precursors into postmitotic neurons which we could then detach from culture plates and successfully freeze down in a viable state. When replated in neuronal medium without neurodifferentiating factors, these ready-to-use human spinal cord neurons remained viable, postmitotic and regenerated neurites in a cell density-dependent manner. Insulin-like growth factor 1 and growth hormone had no effect on neurite regeneration while brain-derived neurotrophic factor increased both the number of cells with neurites as well as the average neurite length. Our model can be applied to investigate factors involved in neuroregeneration of the human spinal cord and since adherent dissociated cell cultures are used, this system has significant potential as a screening platform for therapeutic agents to treat spinal cord injury.     

Acetylcholinesterase neurons in the lateral hypothalamus project to the spinal cord

The possibility that cholinergic neurons of the basal forebrain project to the spinal cord was investigated utilizing the retrograde transport of bisbenzimide and [³H]choline as well as acetylcholinesterase histochemistry. A striking similarity was observed in the distributions of lateral hypothalamic neurons labeled with bisbenzimide and acetylcholinesterase. Only a small number of these neurons contained both markers, suggesting the possibility of a small cholinergic projection from the lateral hypothalamus to the spinal cord. The use of [³H]choline to test this hypothesis yielded negative results. Thus the present observations indicating that lateral hypothalamic neurons are cholineceptive, are consistent with choline acetylcholinesterase immunohistochemical studies. The potential for basal forebrain cholinergic neurons to influence spinal autonomic activity via the lateral hypothalamus is discussed.     

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.