Fibroblast growth factor-induced increased survival of cholinergic mesopontine neurons in culture

Basic fibroblast growth factor (bFGF) was found to increase the survival of immunocytochemically-identified cholinergic mesopontine neurons in dissociated cell cultures of embryonic rat midbrain. In contrast, cultures exposed to, (a) bFGF and an antibody to bFGF, (b) antibody to bFGF alone, or (c) untreated, contained approximately half the number of cholinergic neurons compared to bFGF-treated cultures.     

Development of cholinergic pedunculopontine neurons in vitro: comparison with cholinergic septal cells and response to nerve growth factor, ciliary neuronotrophic factor, and retinoic acid

The well-documented role of nerve growth factor (NGF) in the function of cholinergic neurons in the mammalian basal forebrain can be regarded as a paradigm for the action of trophic substances on CNS neurons. Although several growth factors have been identified in recent years, the specificities and importance of such factors for the development of the nervous system are still unknown. In the present study it has been tested whether NGF affects the group of pedunculopontine cholinergic neurons. This population, which has been described in detail only recently, is located more caudally than but resembles, in some aspects, the basal forebrain cholinergic neurons. The cell bodies are located in the metencephalic pedunculopontine and dorsolateral tegmental nuclei. Similar to the forebrain cholinergic neurons, they are medium to large in size and ascend centrally with long axons. Projection areas are widespread throughout the mesencephalon and diencephalon. Dissociated pontine and septal cells of fetal rat brain (embryo ages E14 to E17) were grown in culture for 7 to 14 days in the presence or absence of NGF. Furthermore, a possible action of retinoic acid and ciliary neuronotrophic factor (CNTF) on cholinergic neurons of both the basal forebrain and the pontine area were tested. Differentiation of cultured cholinergic neurons was assessed by biochemical determination of choline acetyltransferase (ChAT) activity and by immunocytochemical staining for ChAT. NGF in concentrations of 1 to 1,000 ng/ml medium increased the number of immunostained cells and the staining intensity in ChAT immunocytochemistry and enhanced ChAT activity by at least 100% above control levels in septal cultures, thus confirming earlier results. In marked contrast, the same concentrations of NGF failed to influence ChAT activity or immunocytochemical staining in cultures of the pontine area. Retinoic acid (10(-8) M to 10(-5) M) and CNTF (0.2 and 2.0 ng/ml, corresponding to 1 and 10 trophic units, as defined in the ciliary ganglion cell assay) failed to enhance ChAT activity in either culture system and did not potentiate the NGF-mediated increase of ChAT activity in septal cultures. Our results, which indicate that pedunculopontine cholinergic neurons do not respond to NGF during development, are in line with those of NGF-receptor visualization studies that failed to demonstrate such receptors on cholinergic pontine cells in postnatal and adult rats. The findings further underline the specificity of NGF action in the central nervous system and, in particular, do not support the idea of transmitter-specific neurotrophic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cooperative effects of Sonic Hedgehog and NGF on basal forebrain cholinergic neurons

In ventromedial cells of the developing CNS, Sonic hedgehog (Shh) has been shown to affect precursor proliferation, phenotype determination, and survival. Here we show that Shh and its receptor, Ptc-1, are expressed in the adult rat basal forebrain, and that Ptc-1 is expressed specifically by cholinergic neurons. In basal forebrain cultures, Shh was added alone and in combination with nerve growth factor (NGF), and the number of cholinergic neurons was determined by choline acetyltransferase (ChAT) immunocytochemistry. By 8 days in vitro, Shh and NGF show a synergistic effect: the number of ChAT-positive cells after treatment with both factors is increased over untreated cultures or cultures treated with either factor alone. While Shh increases the overall basal level of proliferation, double-labeling of dividing neuronal precursors with [(3)H]thymidine followed by ChAT immunocytochemistry after they mature, demonstrates that the specific increase in cholinergic neurons is not due to this proliferation enhancement. These experiments imply a role for Shh in the development of postmitotic cholinergic neurons and suggest a therapeutic value for Shh in neurodegenerative disease.     

Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.     

Action of furosemide on GABA and glycine currents in rat septal cholinergic neurons in culture

The action of furosemide on GABA- and glycine-induced currents in rat septal cholinergic neurons in culture was examined by use of the whole-cell voltage-clamp technique. Furosemide inhibited the peak amplitude of either GABA or glycine current in a dose-dependent manner; its half-maximal concentration for them was some 2 and 1 mM, respectively. Furosemide at a concentration of 1 mM depressed the maximal peak amplitude of the dose–response curve for either GABA or glycine to about 70% of control. These results indicate that furosemide inhibits in a noncompetitive fashion GABA and glycine receptor-channels in septal neurons.     

Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons

Intraventricular nerve growth factor (NGF) infusion in the adult rat can prevent and also, if delayed, reverse the disappearance of most of the axotomized medial septum cholinergic neurons immunostained for choline acetyltransferase (ChAT). We have utilized the delayed NGF treatment protocol to (i) extend to 3 months the delay time between axotomy and NGF treatment, (ii) define the time course of their recovery, (iii) determine that immunostaining for the (lower affinity) NGF receptor (NGFR) parallels loss and reversal of the ChAT marker, and (iv) evaluate changes in cholinergic somal size following axotomy and subsequent NGF treatment. While NGF treatments starting only 7 days after the fimbria-fornix transection (axotomy) almost entirely restored the number of both ChAT- and NGFR-positive medial septum neurons, longer delayed (2-3 weeks) treatment brought about recovery from the baseline of 20-25% to only about 70% of the control numbers. This limited recoverability, however, persisted even after a 95 day delay period. In all cases examined maximal recoveries were achieved within 3-7 days of NGF treatment. Neuronal size analyses provided evidence for an axotomy-induced atrophy. NGF treatments, started with 1 or 2 week delays, not only reversed fully the average somal size loss but also induced an actual hypertrophy of several of those neurons. These results provide additional evidence that at least half of the apparent loss of cholinergic medial septum neurons upon axotomy is due to a loss of markers such as the transmitter-related enzyme ChAT and NGFR rather than to actual neuronal cell death. These results also show that NGF exerts a genuine trophic influence by regulating the size of its target neurons as well as their content of several proteins.     

Absence of p75(NTR) expression reduces nerve growth factor immunolocalization in cholinergic septal neurons

Septal axons provide a cholinergic innervation to the nerve growth factor (NGF)-producing neurons of the mammalian hippocampus. These cholinergic septal afferents are capable of responding to target-derived NGF because they possess trkA and p75(NTR), the two transmembrane receptors that bind NGF and activate ligand-mediated intracellular signaling. To assess the relative importance of p75(NTR) expression for the responsiveness of cholinergic septal neurons to hippocampally derived NGF, we used three lines of mutant and/or transgenic mice: p75(-/-) mice (having two mutated alleles of the p75(NTR) gene), NGF/p75(+/+) mice (transgenic animals overexpressing NGF within central glial cells and having two normal alleles of the p75(NTR) gene), and NGF/p75(-/-) mice (NGF transgenic animals having two mutated alleles of the p75(NTR) gene). BALB/c and C57B1/6 mice (background strains for the mutant and transgenic lines of mice) were used as controls. Both lines of NGF transgenic mice possess elevated levels of NGF protein in the hippocampus and septal region, irrespective of p75(NTR) expression. BALB/c and C57Bl/6 mice display comparably lower levels of NGF protein in both tissues. Despite differing levels of NGF protein, the ratios of hippocampal to septal NGF levels are similar among BALB/c, C57B1/6, and NGF/p75(+/+) mice. Both p75(-/-) and NGF/p75(-/-) mice, on the other hand, have markedly elevated ratios of NGF protein between these two tissues. The lack of p75(NTR) expression also results in a pronounced absence of NGF immunoreactivity in cholinergic septal neurons of p75(-/-) and NGF/p75(-/-) mice. BALB/c, C57B1/6, and NGF/p75(+/+) mice, on the other hand, display NGF immunoreactivity that appears as discrete granules scattered through the cytoplasm of cholinergic septal neurons. Elevated levels of NGF in the hippocampus and septal region coincide with hypertrophy of cholinergic septal neurons of NGF/p75(+/+) mice but not of NGF/p75(-/-) mice. Levels of choline acetyltransferase (ChAT) enzyme activity are, however, elevated in the septal region and hippocampus of both NGF/p75(+/+) and NGF/p75(-/-) mice, compared with control mice. These data indicate that an absence of functional p75(NTR) expression disrupts the normal cellular immunolocalization of NGF by cholinergic septal neurons but does not affect the ability of these neurons to respond to elevated levels of NGF, as determined by ChAT activity.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture

HSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (>99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (<1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X-100-soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43°C90 min), the level of HSP27 exproion ryas not altered in astrocytes but was elevated significantly in neurons (11–21%) and microglia (4–7%) during 8–48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat-stressed conditions. Following heat shock treatment (45°C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat-stressed conditions, HSP27 was distributed within the Triton X-100-insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and αβ-crystallin were coexpressed in astrocytes under unstressed and heat-stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat-induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous system. © 1995 Wiley-Liss, Inc.     

Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin- insulin-like growth factor-I and thyroid hormone on neuronal survival, growth and differentiation

We have developed a pure cortical neuronal culture free of glial cells, grown in a serum-free environment. The cultured cells immunostained positively with neurofilament antibody while they displayed virtually no glial cell characteristics, such as glial fibrillary acidic protein, glycerol phosphate dehydrogenase or glutamine synthetase. Insulin and transferrin were necessary and sufficient for neuronal survival, neurite extension and glutamic acid decar☐ylase (GAD) expression. Insulin-like growth factor-I was able to replace insulin and was active close to its physiological concentration, suggesting it might be the vivo factor influencing neuronal growth in the brain. The dynamics of the developmental process were striking. The neurons moved on the poly-d-lysine covered plastic dish, and rearrangements in contacts between cells were observed. At first the neurons underwent a general cellular growth manifested by a large increase in the culture total protein content and by the initiation of neurites. A more specific differentiation, as indicated by the sharp increase in GAD levels which was concurrent with an increase in interneuronal contacts, lagged behind the initial growth. Thyroid hormone (TH) affected the differentiation process, causing a future increase in GAD levels during the same time of increase in neurite growth, in interneuronal contacts, in thyroid hormone receptors and thyroid gland maturation. Removal of each of the hormones after a few days of cell growth revealed that transferrin was still required for neuronal survival while insulin became essential for general cellular growth but not specific neuronal differentiation, since it caused an increase in both the total protein and GAD levels but not in GAD specific activity. TH, on the other hand, affected the differentiation process as evident by its ability to increase GAD specific activity. This action of TH, however, required the presence of insulin, without which no increase in GAD level by TH was observed. This neuronal culture, glial and serum-free, provides a new system for investigating neuronal development and function in the complex mammalian central nervous system.     

Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in areas of the brain, including the septal nucleus. Certain growth factors may promote the long-term survival of this subpopulation of neurons at risk. This study was undertaken to characterize growth factors' long-term effects on survival and development of neurons expressing the calcium-binding protein calbindin. In order to accomplish this, embryonic day 16 rat septal neurons were grown in bilaminar culture with astrocytes and in the absence of serum. These cultures were chronically treated with estrogen (Es), insulin-like growth factors I/II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Insulin-like growth factor II significantly increased the number of neurons immunoreactive for calbindin by 155%, suggesting either an increase in the survival of this subpopulation or an increase in the percentage of cells expressing calbindin. Chronic treatment with NGF, IGF-II, and Es significantly increased the number of primary neuritic processes on calbindin-positive neurons, whereas NGF and Es caused significant increases in the number of secondary processes and in the total lengths of the neuritic processes. Thus, effects of IGF-II, estrogen, and NGF on survival and maintenance of this neuronal subpopulation may be dependent on alterations in neurons which are immunopositive for calbindin.     

Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telecephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5–50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5–5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.     

Influence of basic fibroblast growth factor on carbonic anhydrase expression by rat glial cells in primary culture

Modifications of the morphology, the proliferation and the synthesis of carbonic anhydrase of glial cells in primary cultures maintained in defined medium have been investigated under the action of basic fibroblast growth factor. Cultures contained essentially three cell types: astrocytes which expressed glial fibrillary acidic protein, oligodendrocytes which were characterized by the presence of carbonic anhydrase and precursor cells in which these two proteins were detected by immunocytochemistry. In the presence of basic fibroblast growth factor astrocytes and oligodendrocytes underwent morphological changes, characterized by a fibrous aspect; astroglial cells acquired essentially several long processes and oligodendroglial cells formed generally two long processes. The factor increased the proliferation of these two cell types. The quantity of carbonic anhydrase per oligodendrocyte was enhanced in treated cultures. The double-stained precursor cells were present between days 7 and 11 of culture in defined medium, while in the presence of fibroblast growth factor these cells were more numerous and were still present after 14 days. The basic fibroblast growth factor stimulated the proliferation of these young glial cells and modified their morphology. But the differentiation of precursor cells towards one glial cell type appeared to be delayed.     

Rescue of lesioned septal cholinergic neurons by nerve growth factor: specificity and requirement for chronic treatment

We earlier reported that chronic intraventricular injections of NGF into adult rats with partial transection of the fimbria prevent the lesion-induced disappearance of cholinergic neurons in the medial septal nucleus and the diagonal band of Broca (Hefti, 1986). The present study assessed the specificity and treatment requirements of this effect of NGF. Immunohistochemical visualization of NGF receptors (NGF-R) revealed that these molecules are selectively located in forebrain cholinergic neurons of unlesioned brains. Fimbrial transection resulted in transient accumulation of NGF-R in proximal stumps of lesioned axons but failed to induce the expression of NGF-R by other cells in the septal area or near the lesion. Two to three weeks after lesioning, the number of septal neurons expressing NGF-R was reduced by approximately 50% in parallel with the reduction of the number of neurons expressing cholinergic marker enzymes. Repeated intraventricular NGF injections during 4 weeks prevented the disappearance of these cells. Fimbrial transections also reduced the number of septal GABAergic neurons visualized by glutamate decarboxylase immunohistochemistry. The loss of GABAergic neurons was not prevented by NGF. These findings suggest that NGF prevents the lesion-induced degeneration of cholinergic neurons by directly acting on NGF-R expressed by cholinergic cells and that NGF does not affect any neuron with an axonal lesion. Delayed start of the NGF treatment failed to prevent the disappearance of lesioned cholinergic neurons, providing evidence that NGF treatment indeed promotes the survival of these cells rather than simply upregulating the expression of transmitter-specific enzymes. A single injection of NGF at the time of the lesion was not sufficient to prevent the lesion-induced degeneration of cholinergic neurons. Furthermore, termination of chronic NGF treatment after 4 weeks was followed by loss of septal cholinergic neurons after an additional 4 weeks. These findings suggest that the continuous presence of NGF during more than 4 weeks is required to prevent the degeneration of cholinergic cells. The data are discussed in the context of a possible physiological role of NGF in the function of adult forebrain cholinergic neurons.     

Phospholipase C isozymes in neurons and glial cells in culture: an immunocytochemical and immunochemical study

Expression of 3 brain isozymes of phospholipase (PLC-beta, PLC-gamma and PLC-delta) was investigated in relation to cell types found in rat CNS cultures. Immunoreactivity of cultured neurons, astrocytes and oligodendrocytes was demonstrated for all of the 3 isozymes by immunocytochemical staining and immunoblotting, with some differences in reaction intensity. Immunoblotting revealed that the level of expression was neurons greater than oligodendrocytes greater than astrocytes for PLC-beta and PLC-gamma, and astrocytes greater than oligodendrocytes greater than neurons for PLC-delta.     

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.     

Development of cholinergic retinal neurons from embryonic chicken in monolayer cultures: stimulation by glial cell-derived factors

In recent years evidence has indicated that, like the PNS, the development of the CNS is influenced by neuronotrophic polypeptide factors. In the present study, cultures of dissociated retinal neurons from 8-d-old chicken embryos were used to investigate the role of neuronotrophic factors (NTF) in the development of the neural retina. CAT, which in vivo is located in amacrine cells of the retina, served as a marker for studying the in vitro development of cholinergic retinal neurons. Differentiation of cholinergic cells under control conditions was indicated by a 10-fold increase of enzyme activity during a 7-d culture period. Addition of media conditioned by high-density retinal cultures resulted in a further stimulation of CAT activity by 100-400%. The CAT-stimulating activity was associated with a high-molecular-weight component of the retina conditioned medium (RCM) and was sensitive to protease treatment, but was not affected by other hydrolytic enzymes. The putative cholinergic factor was secreted by retinal cultures virtually free of neurons, suggesting that it is mainly produced by Müller cells. CAT-stimulating activity was also present in extracts from embryonic chicken retinae and medium conditioned by rat retinal cultures. NGF, anti-NGF antiserum, extracts from chicken brain tissues, and a number of other extracts and conditioned media, all known to contain neuronotrophic activities, were found to have no influence on cholinergic development in chicken retinal cultures. An extract from non-retinal eye tissue containing ciliary neuronotrophic factor (CNTF) stimulated CAT activity to the same extent as did RCM.(ABSTRACT TRUNCATED AT 250 WORDS)