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The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016.

Neuroendocrine chromaffin cells exist in both intra- and extra-adrenal locations; the organ of Zuckerkandl (OZ) constitutes the largest accumulation of extra-adrenal chromaffin tissue in mammals. The OZ disappears postnatally by modes that are still enigmatic but can be maintained by treatment with glucocorticoids (GC). Whether the response to GC reflects a pharmacological or a physiological role of GC has not been clarified. Using mice with a conditional deletion of the GC-receptor (GR) gene restricted to cells expressing the dopamine β-hydroxylase (DBH) gene [GR(fl/fl) ; DBHCre abbreviated (GR(DBHCre) )], we now present the first evidence for a physiological role of GC signalling in the postnatal maintenance of the OZ: postnatal losses of OZ chromaffin cells in GR(DBHCre) mice are doubled compared to wild-type littermates. We find that postnatal cell loss in the OZ starts at birth and is accompanied by autophagy. Electron microscopy reveals autophagic vacuoles and autophagolysosomes in chromaffin cells. Autophagy in OZ extra-adrenal chromaffin cells is confirmed by showing accumulation of p62 protein, which occurs, when autophagy is blocked by deleting the Atg5 gene (Atg5(DBHCre) mice). Cathepsin-D, a lysosomal marker, is expressed in cells that surround chromaffin cells and are positive for the macrophage marker BM8. Macrophages are relatively more abundant in mice lacking the GR, indicating more robust elimination of degenerating chromaffin cells in GR(DBHCre) mice than in wild-type littermates. In summary, our results indicate that extra-adrenal chromaffin cells in the OZ show signs of autophagy, which accompany their postnatal numerical decline, a process that is controlled by GR signalling.

The LRRK2 gene was recently found to have multiple mutations that are causative for the most common inherited form of late onset Parkinson's disease. In the adult brain, LRRK2 mRNA is broadly expressed, also in regions other than the nigrostriatal system. In order to establish a basis for assessing more detailed functional implications of LRRK2 in development, we provide here an in-depth analysis of its mRNA expression patterns in neural and extra-neural tissues with a focus on murine embryonic development. LRRK2 mRNA is detectable at E8.5 in non-neural and at E10.5 in neural tissues. From E12.5 to E16.5, LRRK2 mRNA is prominently expressed throughout the neocortex and subsequently highly concentrated in ventricular and subventricular zones and cortical plate. In addition, developing cerebellar granule and Purkinje neurons and spinal cord neurons display robust LRRK2 expression. In non-neural tissues LRRK2 was highly expressed in limb interdigital zones, developing kidney glomeruli, and spermatogenetic cells. Together, our results suggest roles for LRRK2 in controlling proliferation, migration, and differentiation of neural cells as well as in morphogenesis of extra-neural tissues.

Extracellular signal-regulated kinase (ERK) is a versatile protein kinase that regulates many cellular functions. Growing evidence suggests that ERK1/2 plays a crucial role in promoting cell death in a variety of neuronal systems, including neurodegenerative diseases. It is believed that the magnitude and the duration of ERK1/2 activity determine its cellular function. In this review, we summarize recent evidence for a role of ERK1/2 in neuronal death. Furthermore, we discuss the mechanisms involved in ERK1/2 mediating neuronal death.

Transforming growth factor-betas (TGF-betas) are among the most widespread and versatile cytokines. Here, we first provide a brief overview of their molecular biology, biochemistry, and signaling. We then review distribution and functions of the three mammalian TGF-beta isoforms, beta1, beta2, and beta3, and their receptors in the developing and adult nervous system. Roles of TGF-betas in the regulation of radial glia, astroglia, oligodendroglia, and microglia are addressed. Finally, we review the current state of knowledge concerning the roles of TGF-betas in controlling neuronal performances, including the regulation of proliferation of neuronal precursors, survival/death decisions, and neuronal differentiation.

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest. Developmental Dynamics 236:3514–3523, 2007. © 2007 Wiley-Liss, Inc.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2 (-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

Evidence is presented that the seminal vesicle of the bull is an important source of a protein which is very similar to nerve growth factor from mouse submaxillary gland. Its chemical and biological properties in vitro are compared with those of nerve growth factor from other sources.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

Declines in memory function and behavioural dysfunction accompany normal ageing in mammals. However, the cellular and morphological basis of this decline remains largely unknown. It was assumed for a long time that cell losses in the hippocampus accompany ageing. However, recent stereological studies have questioned this finding. In addition, the effect of ageing is largely unknown in another key structure of the memory system, the amygdala. In the present study, we have estimated neuronal density and total neuronal numbers as well as density of fragments of degenerated axons in different hippocampal subfields and amygdaloid nuclei. Comparisons were made among aged (21-26 months old) mice and normal adult littermates (8 months old). No significant volume loss occurs in the hippocampus of aged mice. Small but insignificant reductions in total neuronal numbers were found in the hippocampus and in the amygdaloid nuclei. In contrast to the mild effects of ageing upon neuronal numbers, fragments of degenerated axons were increased in both hippocampus and amygdala of aged mice. These data suggest that ageing does not induce prominent cell loss in the hippocampus or amygdala, but leads to degeneration of axons that innervate these forebrain structures. Thus, mechanisms underlying age-related dysfunction depend on parameters other than neuronal numbers, at least in the hippocampal formation and the amygdala.

We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.

Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-beta in terms of its biological activity. The assay used to assess the activity of TGF-beta is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-beta 1, -beta 2, and -beta 3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-alpha, platelet-derived growth factor-AB, insulin-like growth factor-1, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-beta superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain approximately 1 ng of TGF-beta/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-beta 1/beta 2/beta 3. TGF-beta is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10(-5) M). These data suggest that TGF-beta is stored in chromaffin granules and can be released by exocytosis.

The developmental diversification of neural crest-derived sympathoadrenal (SA) progenitor cells into neuroendocrine adrenal chromaffin cells and sympathetic neurons has been thought to be largely understood. Based on two decades of in vitro studies with isolated SA progenitor and chromaffin cells, it was widely assumed that chromaffin cell development crucially depends on glucocorticoid hormones provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor has revealed that the chromaffin cell phenotype develops largely normally in these mice, except for the induction of the adrenaline synthesizing enzyme phenylethylamine N-methyl transferase. In a search for novel candidate genes that might be involved in triggering the sympathetic neuron/chromaffin cell decision, we have studied putative contributions of transforming growth factor (TGF)-alpha, BMP-4, and the transcription factor MASH-1, molecules with distinct expressions in SA progenitor cells, in their migratory pathways and final destinations. TGF-beta2 and -beta3 and BMP-4 are highly expressed in the wall of the dorsal aorta and in the adrenal anlagen during and after immigration of SA progenitors but expressed at much lower levels in sympathetic ganglia. We found that neutralizing antibodies against all three TGF-beta isoforms applied to the chorionic-allantoic membrane (CAM) of quail embryos interfere with proliferation of immigrated adrenal chromaffin cells but do not affect their specific neuroendocrine ultrastructural phenotype. Grafting of noggin-producing cells to the CAM, which scavenges BMPs, interferes with visceral arch and limb development but does not overtly affect the chromaffin phenotype. The transcription factor MASH-1 promotes early differentiation of SA progenitors. Mice deficient for MASH-1 lack sympathetic ganglia, whereas the adrenal medulla previously has been reported to be present. We show here that most adrenal medullary cells in MASH-1(-/-) mice identified by Phox2b immunoreactivity lack the catecholaminergic marker tyrosine hydroxylase. More surprisingly, most cells do not contain chromaffin granules and display a neuroblast-like ultrastructure and show strongly enhanced expression of c-RET comparable to that observed in sympathetic ganglia. Together, our data suggest that TGF-betas and BMP-4 do not seem to be essential for chromaffin cell differentiation. In contrast with previous reports, however, MASH-1 apparently plays a crucial role in chromaffin cell development.

Connexins (cx) constitute a family of transmembrane proteins that form gap junction channels allowing metabolic and electrical coupling of cellular networks. Initial studies on the expression of cx in the developing brain have suggested that cx may undergo dynamic changes and may possibly be implicated in synchronizing development and differentiation of neural progenitor cells and young neurons. We have investigated expression of cx26, cx32, cx43, and cx45 in the midbrain floor, where nigrostriatal dopaminergic neurons originate and differentiate. This neuron population is of major importance in regulating motor-functions. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed low levels of cx26-mRNA in the midbrain floor at E12, which gradually increased during pre- and postnatal development, reaching a maximum in the adult. Cx32-mRNA-levels reached a first peak at E16, and showed highest levels in adulthood. Cx43 was highly expressed at E12, decreased until E18, and subsequently increased again until adulthood. Cx45 mRNA was prominent at all developmental ages, but slightly decreased after the first postnatal week. Double-labeling for the dopaminergic neuronal marker tyrosine hydroxylase (TH), and cx-immunoreactivities (ir) evaluated by quantitative confocal laser microscopy revealed both distinct and similar developmental patterns for the individual cx investigated. Cx26 was highest at E14, decreased towards birth, and subsequently increased again reaching about 50% of the E14 level in the adult. Cx32-ir peaked at E16 and dropped to low levels after birth. Cx43-ir was highest at E12, decreased sharply at E14, reached its lowest levels at birth, but modestly increased again afterwards. Cx45-ir showed a biphasic pattern, with two prominent peaks at E12 and E18, followed by a massive postnatal decrease. Taken together, our results reveal that expression and ir of cx in the midbrain floor and dopaminergic neurons, respectively, follow cx-type specific patterns that temporally coincide with important steps of midbrain morphogenesis, as e.g. progenitor cell formation and migration (E12), early differentiation (E14-16), target encounter (E16-18) and postnatal functional maturation of the nigrostriatal system.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Based on recent evidence from in vitro and gene knockout/insertion studies, this short review summarizes the molecular scenario underlying the development of adrenal chromaffin cells and their preganglionic innervation. During migration of neural crest cells from the dorsal surface of the neural tube to their destinations in the sympathetic primordia and adrenal glands, precursors of the so-called sympathoadrenal (SA) cell lineage are exposed to signals from the notochord and ventral neural tube probably including the protein, Sonic hedgehog. These, and signals in the region of the dorsal aorta (members of the family of bone morphogentic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurones, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells, glucocortiocids have been claimed as essential for suppressing neuronal commitment and for channelling SA cells towards the chromaffin phenotype. However, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth, suggesting that signals other than glucocorticoid hormones may be important in triggering chromaffin cell differentiation. The cholinergic neurones that are preganglionic to adrenal chromaffin cells have their cell bodies located in the intermediolateral column (IML) of the spinal cord. For their normal development, these neurones require signals from the adrenal medulla, which include neurotrophin-4, a major neurotrophic factor of adrenal chromaffin cells. Taken together, these data provide a more complete picture of molecular signalling in the development of one of the most important neuroendocrine tissues in vertebrates.

Chromaffin cells and sympathetic neurons are derivatives of the sympathoadrenal cell lineage of the neural crest. Although they are similar with respect to many cell biological aspects, chromaffin cells, in contrast to sympathetic neurons, continue to synthesize DNA and proliferate through their whole life span. Large numbers of neural and hormonal signals have been implicated in the regulation of chromaffin cell proliferation based on in vitro studies. We have previously shown that chromaffin cells synthesize and release transforming growth factor-beta (TGF-beta) and that exogenously applied TGF-beta suppresses chromaffin cell proliferation in vitro. We show now that TGF-beta is also a physiologically relevant factor in the control of cell division in developing chromaffin cells. We have neutralized endogenous TGF-beta in quail embryos using a monoclonal antibody recognizing all three TGF-beta isoforms, TGF-beta1, -beta2, and -beta3. Embryos deprived of TGF-beta show a prominent increase in numbers of tyrosine hydroxylase (TH)-immunoreactive adrenal chromaffin cells and TH-positive cells incorporating 5'-bromo-2'deoxyuridine. This is the first documentation of the physiological significance of a factor that has been suggested to play a role in the regulation of chromaffin cell mitosis based on in vitro experiments.

Serotonergic neurons in the central nervous system are crucial in the control of autonomic functions and behavior. Mechanisms by which development and maintenance of the serotonergic transmitter phenotype is regulated include activation of protein kinase A (PKA). Using cultures established from the E14 rat raphe we show here that forskolin (10 microM) increases numbers of neurons expressing tryptophan hydroxylase (TpOH), the key enzyme of serotonin synthesis, and uptake of the false serotonergic transmitter 5, 7-dihydroxytryptamine (5,7-DHT). As shown by short-term treatments the effect is due to phenotype induction rather than survival. To begin to understand downstream or parallel signaling pathways required for the PKA-mediated induction of serotonergic markers, we have studied the putative implication of brain-derived neurotrophic factor (BDNF) and its receptor trkB. Treatment of raphe neurons with forskolin induced BDNF mRNA assayed by competitive RT-PCR. Moreover, trkB-IgG receptor bodies fully prevented the forskolin-induced numerical increase in TpOH- and 5,7-DHT-positive cells suggesting an implication of a TrkB-activated pathway. TrkC-IgG had no effect. K252b, a specific inhibitor of trk kinase activity likewise abolished the induction of serotonergic markers by forskolin. In turn, the inductive effect of BDNF on serotonergic markers was blocked by KT5720, a specific inhibitor of PKA. Taken together, these data suggest that co-activation of cAMP- and trkB-dependent signaling pathways plays a crucial role in the regulation of the serotonergic neuronal phenotype.

Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are members of the transforming growth factor-beta superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP-2, -6 and -7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5-HT) neuron development, we have generated serum-free cultures of 5-HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP-6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5, 7-dihydroxytryptamine (5,7-DHT) by about two-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short-term treatments. Importantly, BMP-7 selectively induced 5, 7-DHT uptake without affecting TpOH immunoreactivity. BMP-6 and -7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP-6 and -7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial-derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP-mediated effects on serotonergic development, suggesting that the glia-derived factor is probably brain-derived neurotrophic factor (BDNF) or neurotrophin-4. In support of this notion, we detected increased levels of BDNF mRNA in BMP-treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5-HT neuron development.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.

Serotonergic (5-HT) neurons of the CNS develop as two separate clusters, a rostral and a caudal group, within the brain stem raphe. We show here that the transforming growth factors -β2 and -β3 (TGF-β) and the TGF-β type II receptor are expressed in the embryonic rat raphe, when 5-HT neurons develop and differentiate. To investigate putative roles of TGF-βs in the regulation of 5-HT neuron development we have generated serum-free cultures isolated either from the rostral or the caudal embryonic rat raphe, respectively. In cultures from the caudal E14 raphe saturating concentrations (5 ng/ml) of TGF-β2 and -β3 augmented numbers of tryptophan hydroxylase (TpOH) -immunoreactive neurons and cells specifically taking up 5,7-dihydroxytryptamine (5,7-DHT) by about 1.7-fold over a period of 4 days. Treatment with TGF-βs also increased uptake of 3H- 5HT uptake about 1.7-fold. Alterations in 5-HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of delayed short-term treatments. Comparing rostral and caudal raphe cultures from different embryonic ages suggests that distinct effects of TGF-βs reflect the responsiveness of 5-HT neurons at different ages rather than of different origins. J. Neurosci Res. 56:531–538, 1999. © 1999 Wiley-Liss, Inc.

When fetal calf serum (FCS) alone is used as a trophic support for cultured chicken parasympathetic ciliary ganglionic (cCG) neurons, it does not show any survival-promoting effects on these neurons. When FCS is applied to heparin-affinity chromatography, however, potent survival-promoting activity is obtained in the fraction eluted with 0.5 M NaCl. Using cCG neurons as a bioassay system, this neurotrophic activity was purified by a combination of heparin-affinity chromatography, gel filtration chromatography, and Sep-Pak C18 cartridge. The 40-50-kDa fractions from the gel filtration column with strong survival-promoting activity were shown to contain insulin-like growth factor II (IGF-II) by immunoblot analysis. By acidification, the survival-promoting activity and IGF-II were translocated together from the 40-50-kDa to the 7-10-kDa fractions, and the survival-promoting activity in the 7-10-kDa fractions was blocked by an anti-IGF-II neutralizing monoclonal antibody. These results indicate that the neurotrophic substance in 0.5 M NaCl-eluate from heparin-affinity chromatography is IGF-II and that mechanisms may exist in vivo for the activation of latent IGF-II, whose biological effects may be blocked by its specific binding proteins.

We investigated putative roles of transforming growth factor (TGF)-beta expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-beta2 and -beta3 as well as the TGF-beta receptor TbetaR-II, but are not ir for TGF-beta1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-beta3 mRNA and TGF-beta biological activity in cultures of E8 CG neurons. None of the TGF-beta isoforms--beta1, beta2, or beta3--has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-beta significantly promotes CG neuron survival. However, TGF-beta does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-beta released from CG neurons using an antibody to TGF-beta1/-beta2/-beta3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti-pan-TGF-beta antibody could be rescued by adding exogenous TGF-beta. Together, these data suggest that para-/autocrine TGF-beta signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons.

Astrocytes are coupled by gap junctions, which are composed of connexins, channel-forming proteins. Connexin43 is the predominant connexin of astrocytes. Gap junctions assemble astrocytes into functional syncytia permitting exchange of small molecules including metabolites, catabolites, and second messenger molecules. Thus, gap junctions of astroglial cells serve the maintenance of extra- and intra-cellular homeostasis in the brain and eventually ascertain neuronal functions. Alterations in astroglial cell coupling can disturb this equilibrium resulting in neuronal dysfunction and death. Growth factors are an important class of substances that can influence coupling in non-neural cells. Several groups have recently carried the analyses of gap junction regulation by cytokines to the level of neural cells. This review summarizes the recent progress in this field and outlines directions of future research.

Fibroblast growth factor (FGF)-2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation-promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)-β, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF-2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF-β that neutralize the isoforms -β1, -β2 and -β3 abolish the trophic effect of FGF-2. FGF-2 increases TGF-β3 mRNA and amounts of biologically active TGF-β determined in a mink lung epithelial cell assay in a time-dependent manner. FGF-2 also induces levels of active TGF-β in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF-β is required for mediating the survival promoting effect of FGF-2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.

Fibroblast growth factor (FGF)-2 is a peptide growth factor that promotes the generation, differentiation, and survival of neurons and glial cells. In the CNS, astroglial cells are coupled in a region-specific manner by gap junctions consisting of connexin 43 (cx43). In the present study we have investigated effects of FGF-2 and of other growth factors on the expression and function of cx43 in astroglial cells cultured from telencephalic cortex, striatum, and mesencephalon of newborn rats. Confluent cultures were maintained for two days in low serum, and then exposed to FGF-2 (10 ng/ml) for 48 h. FGF-2 caused a reduction of cx43-protein, -mRNA, and intercellular communication revealed by dye spreading. These changes occurred in cortical and striatal cells, but not in mesencephalic astroglial cells. Effects of FGF-2 were time- and concentration-dependent, with a minimal effective dose of 1 ng/ml FGF-2, and an onset of effects after 6 h of incubation. The reduction of coupling by FGF-2 was transient, since in cortical and striatal cultures coupling recovered to control levels 48 h after removal of the growth factor. Like FGF-2, transforming growth factor-beta3 (TGF-beta3) decreased coupling of cortical and striatal, but not mesencephalic astroglial cells. Astroglial cells from all brain regions showed a slight FGF-mediated increase in 5-bromo-2'-desoxy-uridine (BrdU) incorporation, which was abolished upon co-treatment with TGF-beta3. However, TGF-beta3 did not interfere with the repression of cx43-function by FGF-2. Epidermal growth factor (EGF) that has been demonstrated to influence coupling in other cell types had no effect on dye spreading but significantly increased BrdU incorporation. Our results reveal a novel function of FGF-2 on cultured astroglial cells which may be relevant to the regulation of astroglial cell connectivity in vivo.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta (TGF-beta) superfamily, is a potent neurotrophic factor for several neuron populations in the central and peripheral nervous system. Members of the neurotrophin, neurokine, and TGF-beta families of growth factors can affect neurons beyond their capacity to promote survival. They can play instructive roles including the determination of a particular transmitter phenotype. Here, we show that GDNF enhances the number of calretinin (CaR)-positive neurons in serum-free cultures of striatal cells isolated from embryonic rats. The effect is dose-dependent, can be elicited with concentrations as low as 0.1 ng/ml, and is not accompanied by increased incorporation of 5-bromo-2'-desoxyuridine and appearance of glial fibrillary acidic protein-positive cells. Similar, but weaker effects can be elicited by brain-derived neurotrophic factor, neurotrophin-3 and -4, fibroblast growth factor-2. Ciliary neurotrophic factor, nerve growth factor, and TGF-beta 1 do not affect striatal CaR expression. GDNF can augment CaR-positive cells at any time point and with a minimal exposure of 18 hr, suggesting induction of the phenotype rather than increased survival. By reverse transcription polymerase chain reaction (RT-PCR), we show that GDNF is expressed in the E16 striatum and in cultures derived from this tissue. GDNF also protected striatal CaR-positive neurons against glutamate toxicity. We conclude that striatal GDNF, in addition to its retrograde trophic role for nigrostriatal dopaminergic neurons, may also act locally within the striatum (e.g., by inducing the CaR phenotype and protecting these cells against toxic insult).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily that have been implicated in tissue growth and remodelling. Recent evidence suggests that several BMPs are expressed in the developing and adult brain. Specifically, we show that BMP 2 and BMP 6 are expressed in the developing midbrain floor of the rat. We studied potential neurotrophic effects of BMPs on the in vitro survival, transmitter uptake and protection against MPP+ toxicity of mesencephalic dopaminergic neurons cultured from the embryonic midbrain floor at embryonic day (E) 14. At 10 ng/ml and under serum-free conditions, most BMPs promoted the survival of dopaminergic neurons visualized by tyrosine hydroxylase immunocytochemistry during an 8-day culture period, but to varying extents (relative potencies: BMP 6 = 12 > 2, 4, 7). BMPs 6 and 12 were as effective as fibroblast growth factor-2 (FGF-2) and glial cell line-derived neurotrophic factor, promoting survival 1.7-fold compared with controls. BMPs 9 and 11 were not effective. Dose-response curves revealed an EC50 for BMPs 2, 6 and 12 of 2 ng/ml. BMPs 2, 4, 6, 7, 9 and 12 also promoted DNA synthesis and astroglial cell differentiation, visualized by 5-bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic protein (GFAP) immunocytochemistry respectively. Suppression of cell proliferation and subsequent maturation of GFAP-positive cells by 5-fluorodeoxyuridine or aminoadipic acid abolished the neuron survival-promoting effect of BMP 2. This suggests that BMPs, like other non-TGF-beta factors affecting dopaminergic neuron survival, act indirectly, probably by stimulating the synthesis and/or release of glial-derived trophic factors. BMP 6 and BMP 7 also increased the uptake of [3H]dopamine without affecting the uptake of [3H]5-hydroxytryptamine and [3H]GABA, underscoring the specificity of the trophic effect. We conclude that several BMPs share a neurotrophic capacity for dopaminergic midbrain neurons with other members of the TGF-beta superfamily, but act indirectly, possibly through glial cells.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote survival and differentiation of midbrain dopaminergic (DAergic) neurons in vitro and in vivo. This is consistent with their expression and that of their cognate receptors, trkB and trkC, in the nigrostriatal system. Degeneration of DAergic neurons of the substantia nigra and alpha-synuclein-positive aggregates in the remaining substantia nigra (SN) neurons are hallmarks of Parkinson's disease (PD). Reduced expression of BDNF has been reported in the SN from PD patients. Moreover, mutations in the BDNF gene have been found to play a role in the development of familial PD. We show now that haploinsufficiencies of the neurotrophin receptors trkB and/or trkC cause a reduction in numbers of SN neurons in aged (21-23 month old) mice, which is accompanied by a reduced density in striatal tyrosine hydroxylase immunoreactive (TH-ir) fibers. These aged mutant mice, in contrast to wild-type littermates, display an accumulation of alpha-synuclein in the remaining TH-positive neurons of the SN. We conclude that impairment in trkB and/or trkC signaling induces a phenotype in the aged SN, which includes two hallmarks of PD, losses of TH positive neurons and axons along with massive neuronal deposits of alpha-synuclein.

Basic fibroblast growth factor (bFGF; FGF-2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF-2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF-2, and to analyse in more detail the changes within astroglial cells in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned striatum, we have studied striatal expression and regulation of connexin-43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF-2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF-2 mRNA, GFAP-positive cells, and cx43-immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF-2 in a piece of gelfoam caused a further increase in cx43-positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the non-trophic control protein in cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF-2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF-2 in the MPTP-model of Parkinson's disease. © 1996 Wiley-Liss, Inc.

We have studied the localizations of transforming growth factor-beta (TGF-beta) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-beta 3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-beta 3 mRNA as well as TGF-beta 2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-beta 2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-beta 2 and 3 until E11, but even then the moderate signals for TGF-beta 3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-beta 3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-beta receptor type II, and treatment of these cells with NGF induces expression of TGF-beta 3 mRNA. The TGF-beta isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-beta isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-beta 2 and -beta 3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-beta s in fundamental events of neural development.

Transforming growth factors beta (TGF-beta), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-beta, we have localized the TGF-beta isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-beta 2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-10 and can be selectively marked by NADPH-diaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 micrograms TGF-beta 2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of [125I]TGF-beta 2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-beta may exert its neuroprotective actions by indirect mechanisms. TGF-beta applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-beta exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-beta may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. We show here that GDF5 is expresed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-beta 3, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-beta and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-beta and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD.

Growth factors can induce both proliferation or differentiation of neuroblastoma (NB) cells through interaction with specific receptors. Using two automated colorimetric assays for determinations of cell numbers, the present study demonstrates that (a) different NB and neuroepithelioma cell lines show distinct responses, both qualitatively and quantitatively, to basic FGF (bFGF), NGF, and EGF(b) even closely related NB cell lines (e.g., SK-N-SH, SH-SY5Y, and SHEP) do not respond uniformly to these factors; c) responses of the two neuroepithelioma cell lines employed (SK-N-MC and CHP-100) differ, but match those of certain NB cell lines; and d) two growth factors, bFGF and EGF, may both stimulate or inhibit proliferation, depending on the cell line studied. Specifically, IMR-32, SK-N-SH, and SH-SY5Y showed a mitogenic response to each growth factor. Maximal proliferative responses ranged from 204–355% as compared to controls (100%). GICAN was stimulated by NGF (199%), and SK-N-MC and NMB by EGF (282 and 140%, respectively), but other factors were ineffective. CHP-100 and GIMEN were inhibited by bFGF. NGF and EGF were not effective on CHP-100 cells, while EGF caused an arrest of mitogenic activity in GIMEN cells, and NGF stimulated their proliferation. Cell lines SHEP and LAN1 did not respond to any factor. To begin to analyze putative relationships of growth factor responsiveness and growth factor/growth factor receptor expressions, IMR-32, GIMEN, and LAN1 cell lines were studied for the presence of bFGF, NGF, FGF receptors (R)-1 (flg) and FGFR-4, trk, and low-affinity NGF receptor (p75) mRNAs. All three cell lines expressed bFGF and NGF mRNA, but not the FGFR-1, FGFR-4, trk, and p75 mRNAs. These results suggest extremely diverse patterns of NB/neuroepithelioma cell responsiveness to “mitogenic” growth factors and no overt correlation between such responses and growth factor/growth factor receptor expression. © 1995 Wiley-Liss, Inc.

The superfamily of transforming growth factors-beta (TGF-beta) comprises an expanding list of multifunctional proteins serving as regulators of cell proliferation and differentiation. Prominent members of this family include the TGF-beta s 1-5, activins, bone morphogenetic proteins and a recently discovered glial cell line-derived neurotrophic factor (GDNF). In the present study we demonstrate and compare the survival promoting and neuroprotective effects of TGF-beta 1, -2 and -3, activin A and GDNF for midbrain dopaminergic neurons in vitro. All proteins increase the survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the embryonic day (E) 14 rat mesencephalon floor to varying extents (TGF-beta s 2.5-fold, activin A and GDNF 1.6-fold). TGF-beta s, activin A and GDNF did not augment numbers of very rarely observed astroglial cells visualized by using antibodies to glial fibrillary acidic protein and had no effect on cell proliferation monitored by incorporation of BrdU. TGF-beta 1 and activin A protected dopaminergic neurons against N-methyl-4-phenylpiridinium ion toxicity. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TGF-beta 2 mRNA, but not GDNF mRNA, is expressed in the E14 rat midbrain floor and in mesencephalic cultures. We conclude that TGF-beta s 1-3, activin A and GDNF share a neurotrophic capacity for developing dopaminergic neurons, which is not mediated by astroglial cells and not accompanied by an increase in cell proliferation.

The transforming growth factors beta (TGF-beta), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-beta s, and since TGF-beta mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regeneration) or transfected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-beta 1-3 mRNA were determined for each segment. We demonstrate that TGF-beta 1 mRNA levels are higher than those of TGF-beta 3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-beta 2 mRNA could not be detected. For TGF-beta 1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-beta immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-beta-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-beta-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-beta-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-beta expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-beta mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a "reactive" Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-beta expression.

Based on our previous observations that neuroblastoma (NB) cells express fibroblast growth factor-2 (FGF-2; basic FGF) and respond to it [Janet T. et al. (submitted); Wewetzer K. et al. (1993) J. Neurosci. Res. 36, 209-215), we attempted to find to what extent selected cytokines [interleukin (IL)-1 beta and interferon gamma (IFN gamma)] may modulate FGF-mediated proliferative activity and differentiation. The NB cell lines IMR-32, SH-SY5Y, GIMEN and LAN-1 and colorimetric assays were used for the determination of cell numbers. IL-1 beta (and several other ILs, including IL-1 alpha, -2, -3, and 6) per se did not affect proliferation of any cell line studied. IFN gamma inhibited growth of GIMEN and LAN-1 cells, but was uneffective on IMR-32 and SH-SY5Y cells. FGF-2 was antimitogenic for GIMEN cells. IFN gamma reversed and IL-1 beta enhanced this antimitogenic effect of FGF-2. FGF-2 per se did not affect LAN-1 cells and did not modulate the growth inhibitory actions of IFN gamma on these cells. FGF-2 induced proliferation of IMR-32 and SH-SY5Y cells. This effect was not modulated by IFN gamma or IL-1 beta. These results suggest a heterogeneous response pattern of human NB cell lines towards the cytokines studied and complex interactions of FGF-2, IL-1 beta and IFN gamma.

The autonomic nervous system consists of sympathetic, parasympathetic, and enteric ganglia and also of a specialized neuroendocrine derivative, the adrenomedullary chromaffin cells.

Neuroblastoma cells have been shown to contain growth factors, which may be involved in the regulation of cell proliferation and/or differentiation. We have examined whether human IMR-32 neuroblastoma cells store factors with a capacity to promote neuron survival and differentiation. Heparin affinity chromatography, cell blotting, and isoelectric focusing of IMR-32 cell extracts revealed multiple neurotrophic activities at molecular weights of 16.8, 24.1, 39.0, 45.3, 52.3 and isoelectric points of 4.0, 5.0, 8.3, 9.0. Immunocytochemistry, immunoblotting, and radio-immunoassay with specific antibodies suggests that one neurotrophic activity is immunologically identical with basic fibroblast growth factor (bFGF). This assumption is supported by Northern blot analysis, which shows a 6.0 kb bFGF transcript.

Following a previous study in which we showed ameliorative effects of basic fibroblast growth factor (FGF-2) locally applied to the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice, we investigated FGF-2 actions at different time intervals after the lesion and effects on non-dopaminergic striatal transmitter systems. A triple intraperitoneal injection of 30 mg/kg MPTP at 24 h intervals caused a reduction of striatal dopamine to 23% of control levels that lasted for at least 4 weeks. Four micrograms FGF-2 soaked into gel foam and placed onto the right striatum partially and bilaterally restored dopamine levels and tyrosine hydroxylase activity after 2 weeks, when the treatment started simultaneously or 1 day after the toxin lesion. FGF-2 was ineffective, if administration commenced with a delay of 7 days. Striatal neurotransmitters that are known to be linked to the dopaminergic system were also altered by the MPTP treatment. GABA was significantly increased, while somatostatin levels were reduced. Upon FGF-2 administration both GABA and somatostatin levels were partially normalized. Our data are consistent with the notion that FGF-2 protects and rescues acutely and subacutely MPTP-lesioned nigrostriatal neurons and that its effects must be mainly indirect. Likewise, positive effects of FGF-2 on non-dopaminergic neurons may be due to the partial restoration of striatal dopamine.

To further clarify the function of basic fibroblast growth factor (bFGF) in the nervous system, we have examined its distribution, lesion-dependent regulation, retrograde transport, and trophic roles on rat hypoglossal neurons. In adult rats, bFGF-like immunoreactivity is localized in hypoglossal motoneurons, drastically reduced 2 days after axotomy, and re-expressed by 11 days. Neuron numbers and morphology assessed by Nissl staining are not affected by the lesion. 125J bFGF is specifically retrogradely transported by hypoglossal motoneurons from their peripheral nerve terminals. Moreover, bFGF stimulates the in vitro survival of hypoglossal neurons (ED50 2 ng/ml). In vivo administration of bFGF prevents lesion-induced motoneuron death to 14% in 7 day old rats and to 60% in 18 day old rats, but not the axotomy-induced decrease of choline acetyltransferase activity in the hypoglossal nucleus of adult rats. These results are consistent with a neurotrophic role of bFGF in the hypoglossal system. © 1992 Wiley-Liss, Inc.

We have raised a monoclonal antibody (MAB-1E10) reactive with the intact forms but not the processing products of the chromaffin cell vesicle protein chromogranin B (CgB). The antibody recognizes rat and human, but not bovine and chick adrenal chromaffin cells. In addition, MAB-1E10 immunoreactivity was detected in rat PC 12 pheochromocytoma cells and in pituitaries. Several other tissues, including pancreas, small intestine and superior cervical ganglia, which are known to contain CgB in endocrine cells or neurons, respectively, were found not to be reactive with MAB-1E10. Using short-term cultures of dissociated adrenal chromaffin cells from Hannover-Wistar rats, we found that the expression of intact CgB is developmentally regulated. Between embryonic day 19 and postnatal day 40, about 80% of adrenal chromaffin cells--identified by their reactivity with an antibody against the enzyme dopamine-beta-hydroxylase--were found to be reactive with MAB-1E10. The proportion of positive cells subsequently decreased to about 5% at postnatal day 90. In the presence of glucocorticoids, this decrease was reduced to about 45% CgB-positive cells at postnatal day 90. In another rat strain, Sprague-Dawley rats, the proportion of MAB-1E10-immunoreactive chromaffin cells (about 50%) remained constant from birth to adulthood. Our results indicate that CgB is differentially expressed and/or processed in different rat tissues, strains and during development, and furthermore, that expression or processing in rat chromaffin cells might be regulated by glucocorticoids. Intact CgB appears to be a marker for a subpopulation of chromaffin cells, but its function(s) remains to be clarified.

Chromaffin granules, the secretory organelles of the neuron-like adrenal medullary chromaffin cells, have previously been shown to store and liberate neurotrophic activities that support in vitro survival of several neuron populations including those innervating the adrenal medulla. Molecules resembling fibroblast growth factor and ciliary neurotrophic factor have been identified among these activities. Since chromaffin granules store a variety of neuropeptides and many neuropeptides can have pleiotropic effects on neuronal growth and maintenance we have tested 24 different neuropeptides for their capacities to promote survival of embryonic chick ciliary, dorsal root and sympathetic ganglionic neurons. Peptides tested included several derivatives of proenkephalin (Leu- and met-enkephalin, fragments BAM 22, B, F and E), somatostatin, substance P, neuropeptide Y, neurotensin, VIP, bombesin, secretin, pancreastatin, dynorphin B, dynorphin 1–13, β-endorphin, alpha-, beta-, and gamma-MSH. Control cultures received saturating concentrations of ciliary neurotrophic or nerve growth factor (CNTF; NGF), or no trophic supplements. At 1 × 10 −5 M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leuenkephalin, the proenkephalin fragments BAM 22 and E, and somatostatin maintained about half of the dorsal root ganglionic neurons supported by NGF, but were not effective on ciliary neurons. VIP promoted the survival of approximately 50% of the ciliary and embryonic day 10 dorsal root ganglionic neurons as compared to saturating amounts of CNTF, but required the presence of non-neuronal cells in the cultures to be effective. Neurotensin (1 × 10 −5 M ) had a small effect on ciliary neurons. All other peptides had no effect on any neuron population tested. Our results suggest that neuropeptides that occur in the adrenal medulla may play a role for the maintenance of cholinergic, sensory, and sympathetic neurons innervating the adrenal medulla.

Although a variety of in vitro and in vivo actions of basic fibroblast growth factor (bFGF) on neuronal cells have been documented, the physiological role of this protein in the nervous system is still contested. Since the distribution of a molecule in the nervous system may provide cues for an understanding of its possible roles, we have begun to study its cellular localization in the central and peripheral nervous system using immunocytochemistry with an anti-bFGF-specific antibody. Here we provide an account on the distribution of bFGF-like immunoreactivity (bFGF-IR) in the brainstem of the developing and adult rat. Basic FGF-IR was found to be widely distributed in motor and sensory nuclei. In all nuclei examined, only subpopulations of neurons were stained. Different staining patterns were found. For example, in the red nucleus weakly or unstained perikarya were surrounded by numerous immunoreactive fibers, often in close contact with the neuronal surface. In the reticular formation and facial nerve, many neuronal cell bodies showed a strong IR that extended into the processes. Glial cells were consistently unstained. During early postnatal development changes of the distribution of bFGF IR were found. From this wide distribution pattern of bFGF-IR, we conclude that bFGF may have more general and, possibly, diverse functions rather than a restricted role for a particular subset of neurons. Variations in the staining pattern of nerve cell bodies in a single nucleus may suggest a function related to neuronal activity.

The effect of growth factors with neurotrophic properties on proliferation of the human IMR 32 neuroblastoma (NB) cell line was studied. A colorimetric proliferation assay and an anchorage-dependent cell culture system were used. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), neurite-inducing factor (NIF), ciliary neurotrophic factor (CNTF), and a cell-free extract from selected embryonic chick eye tissues (CIPE) were assayed for their capacity to control proliferation. Basic FGF, NGF, and CIPE stimulated proliferation of IMR 32 NB cells in serum-containing culture conditions. The NIF and CNTF had no effect. The concentration of bFGF required to induce half-maximal cell growth was 4.6 +/- 1.8 ng/ml, but the half-maximally effective dose of NGF was 7.5 +/- 2.7 ng/ml. In combination these two growth factors were additive within a small concentration range. In serum-free culture conditions bFGF affected both proliferation and cell differentiation by promoting neurite growth and cell aggregate formation. In contrast, NGF induced cell neurite outgrowth only. These results, in conjunction with the evidence that bFGF-like molecules are present, in IMR 32 NB cells may support the notion that NB cells regulate their proliferation by an autocrine mechanism. Basic FGF and NGF, two distinct neurotrophic factors, appear to be involved in the regulation of NB cell proliferation.

We have previously shown that basic fibroblast growth factor (bFGF; 2 microg), when administered in gelfoams implanted to medullectomized adult rat adrenal glands, maintains target-deprived preganglionic neurons located in the intermediolateral column (IML) of the spinal cord. We show now that administration of 600 or 60 ng/implant/organ of the biologically active recombinant protein also maintains IML-neurons. The effects depend on an intact organ nerve supply since administration of bFGF-implants to the medullectomized and denervated (splanchnicotomized) organ abolishes the maintenance effects for IML-neurons. A possible regeneration of cholinergic nerve fibres was assessed by quantitative determination of choline acetyltransferase (ChAT)-activity and ultrastructurally under various treatments. When compared to a cytochrome-(Cyto)-C treatment, bFGF significantly increased ChAT-activity in the innervated adrenal gland after 4 weeks, suggesting that sprouting of cholinergic nerve fibres and/or recovery of enzyme activity had occurred. Electron microscopically, unmyelinated axons associated with Schwann cells were only found in bFGF, but not in Cyto-C-implants. We conclude that exogenous bFGF or a yet unidentified trophic molecule possibly induced by bFGF in the adrenal gland acts as a retrogradely transported messenger serving for the maintenance of spinal cord IML-neurons in vivo. By stimulating axonal sprouting, bFGF might facilitate access of trophic material to nerve fibres.

Basic fibroblast growth factor (bFGF) is a potent mitogen for several types of cells, including glial cells, which also seem to express bFGF. We have used rat C6 glioma cells as a model system to study the expression and release of bFGF by glioma cells, as well as the effects of exogenous bFGF on these cells. We have shown that C6 cells express 18 kD bFGF and several higher molecular weight immunoreactive forms. The expression of bFGF could be induced by a factor present in fetal calf serum. Subsequent to its initial appearance, bFGF is regulated in a cell density-dependent manner. Neither bFGF-like immunoreactive material, nor bFGF-like neurotrophic activity were found to be released by C6 cells. Exogenously applied bFGF changed C6 cell morphology similar to cyclic AMP induced alterations but had no significant influence on C6 cell proliferation and biochemical differentiation. From these results we conclude that bFGF in C6 cells might act as an endogenous (not autocrine) mitogen. Possible roles for bFGF in glial cells are discussed.