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Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the “death” phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)–, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3–independent mechanism.

The generation of complex neuronal structures, such as the neocortex, requires accurate positioning of neurons and glia within the structure, followed by differentiation, formation of neuronal connections, and myelination. To understand the importance of TrkB signaling during these events, we have used conditional and knockin mutagenesis of the TrkB neurotrophin receptor, and we now show that this tyrosine kinase receptor, through docking sites for the Shc/FRS2 adaptors and phospholipase Cgamma (PLCgamma), coordinates these events in the cerebral cortex by (1) controlling cortical stratification through the timing of neuronal migration during cortex formation, and (2) regulating both neuronal and oligodendrocyte differentiation. These results provide genetic evidence that TrkB regulates important functions throughout the formation of the cerebral cortex via recruitment of the Shc/FRS2 adaptors and PLCgamma.

FGF-2, a potent multifunctional and neurotrophic growth factor, is widely expressed in the brain and upregulated in cerebral ischemia. Previous studies have shown that intraventricularly or systemically administered FGF-2 reduces the size of cerebral infarcts. Whether endogenous FGF-2 is beneficial for the outcome of cerebral ischemia has not been investigated. We have used mice with a null mutation of the fgf2 gene to explore the relevance of endogenous FGF-2 in brain ischemia. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCAO). We found a 75% increase in infarct volume in fgf2 knock-out mice versus wild type littermates (P < 0.05). This difference in the extent of ischemic damage was observed after 24 h, and correlated with decreased viability in fgf2 mutant mice following MCA occlusion. Increased infarct volume in fgf2 null mice was associated with a loss of induction in hippocampal BDNF and trkB mRNA expression. These findings indicate that signaling through trkB may contribute to ameliorating brain damage following ischemia and that bdnf and trkB may be target genes of FGF-2. Together, our data provide the first evidence that endogenous FGF-2 is important in coping with ischemic brain damage suggesting fgf2 as one crucial target gene for new therapeutic strategies in brain ischemia.

Development of midbrain dopaminergic neurons is known to depend on inductive signals derived from the ventral midline, including Sonic hedgehog (Shh) as one of the identified molecules. Here we show that in addition to Shh, transforming growth factor (TGF)-β is required for both induction and survival of ventrally located midbrain dopaminergic neurons. Like Shh, TGF-β is expressed in early embryonic structures such as notochord and floor plate, as well as in the area where mibrain dopaminergic neurons are developing. Treatment of cells dissociated from the rat embryonic day (E) 12 mibrain floor with TGF-β significantly increases the number of tyrosine hydroxylase (TH)-positive dopaminergic neurons within 24 hr. Neutralization of TGF-βin vitrocompletely abolishes the induction of dopaminergic neurons. In the absence of TGF-β, Shh cannot induce TH-positive neurons, and vice versa, neutralizing endogenous Shh abolishes the capacity of TGF-β to induce dopaminergic neuronsin vitro. Furthermore, neutralization of TGF-βin vivoduring chick E2–7 but not E4–7 resulted in a significant reduction in TH-positive neurons in the ventral midbrain floor but not in the locus coeruleus or diencephalon, which suggests that the TGF-β is required for the induction of mesencephalic dopaminergic neurons with a critical time period at E2/E3. Furthermore, neutralization of TGF-β between E6 and 10, a time period during maturation of mesencephalic dopaminergic neurons when no further inductive cues are required, also resulted in a significant loss of dopaminergic neurons, suggesting that TGF-β is required for the promotion of survival of ventral midbrain dopaminergic neurons as well. Together, our results identify TGF-β as an essential mediator for the induction and maintenance of midbrain dopaminergic neurons.

Transforming growth factor beta2 (TGF-beta2), a prototypic member of a large superfamily of multifunctional cytokines, is expressed by neurons and glial cells. Its subcellular compartmentalization and release from neurons, however, are largely unknown. Here we show that TGF-beta2 colocalizes with the trans-Golgi network marker TGN38 and a marker molecule for secretory granules, chromogranin B (CgB), in PC12 cells. Similarly, primary hippocampal neurons show colocalization of TGN38 and TGF-beta2. A substantial amount of endogenous as well as transfected TGF-beta2 in PC12 cells comigrates with CgB on an equilibrium gradient, suggesting costorage in secretory granules. TGF-beta biological activity peaks in identical fractions. Depolarization of PC12 cells with high potassium triggers colocalization of CgB and TGF-beta2 at the cell surface, suggesting their regulated corelease from secretory granules. High potassium also liberates biologically active TGF-beta from PC12 cells and primary neurons. Our results indicate that a substantial portion of TGF-beta2 is secreted by the regulated secretory pathway in PC12 cells and hippocampal neurons.

Transforming growth factor-betas (TGF-betas) are a still growing superfamily of cytokines with widespread distribution and diverse biological functions. They fall into several subfamilies including the TGF-betas 1, 2, and 3, the bone morphogenetic proteins (BMPs), the growth/differentiation factors (GDFs), activins and inhibins, and the members of the glial cell line-derived neurotrophic factor family. Following a brief description of their general roles and signaling in development, maintenance of homeostasis, and disease, we shall focus on their distribution in the CNS and their involvement in regulating neuron survival and death.

Growth differentiation factor-15 (GDF-15) is a novel member of the transforming growth factor-beta superfamily and has been shown to be induced in neurons subsequent to lesions. We have therefore begun to study its putative role in the regulation of neuron survival and apoptosis. Cultured cerebellar granule neurons (CGN) survive when maintained in high K(+) (25 mm) but undergo apoptosis when switched to low K(+) (5 mm). GDF-15 prevented death of CGN in low K(+). This effect could be blocked by phosphatidylinositol 3-kinase/Akt pathway inhibitors LY294002 or wortmannin. In contrast, mitogen-activated protein kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway inhibitors U0126 and PD98059 potentiated GDF-15 mediated survival and prevented cell death in low K(+) even without factor treatment. Immunoblots revealed GDF-15-induced phosphorylation of Akt and glycogen synthase kinase-3beta. This activation was suppressed by phosphatidylinositol 3-kinase inhibitors. Low K(+) induced delayed and persistent ERK activation, which was blocked by MEK inhibitors or GDF-15. ERK activation induced c-Jun, a member of the AP-1 transcription factor family. GDF-15 or U0126 prevented c-Jun activation. Furthermore, we show that GDF-15 prevented generation of reactive oxygen species, a known activator of ERK. Together, our data suggest that GDF-15 prevents apoptosis in CGN by activating Akt and inhibiting endogenously active ERK.

This review summarizes the evidence that GDF-15, a recently discovered member of the TGF-β superfamily, is a trophic factor for nigral dopamine neurons, both in vitro and in vivo. Specifically, GDF-15 promotes survival and differentiation of embryonic rat dopaminergic neurons, but not of other neuron populations, with the exception of serotonergic raphe neurons. The neurotrophic effect of GDF-15 seems to be direct and not mediated through glial cells. In the rat 6-hydroxydopamine model of parkinsonism GDF-15 rescues intoxicated dopaminergic neurons and abolishes abnormal turning behavior. The most prominent site of synthesis of GDF-15 within the brain is the choroid plexus, which secretes GDF-15 into the cerebrospinal fluid, from where the molecule can penetrate through the ependymal layer into the parenchyma. Analysis of mouse mutants lacking GDF-15 will reveal whether the endogenous factor also has a role in promoting embryonic and protecting lesioned nigral dopamine neurons.

Transforming growth factor-ss (TGF-ss) are a still growing superfamily of cytokines with widespread distribution and diverse biological functions. They fall into several subfamilies including the TGF-ss 1, 2, and 3, the bone morphogenetic proteins (BMPs), the growth/differentiation factors (GDFs), activins and inhibins, and the members of the glial cell line-derived neurotrophic factor family. Following a brief description of their general roles and signaling in development, maintenance of homeostasis, and disease, we shall focus on their distribution in the CNS and their involvement in regulating neuron survival and death.

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.

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.

Transforming growth factor-betas (TGF-betas) constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation, and tissue remodeling. In the developing nervous system, TGF-beta2 and -beta3 occur in radial and astroglial cells as well as in many populations of postmitotic, differentiating neurons. TGF-beta1 is restricted to the choroid plexus and meninges. In addition to functions related to glial cell maturation and performances, TGF-beta2 and -beta3 are important regulators of neuron survival. In contrast to neurotrophic factors, as for example, neurotrophins, TGF-betas are most likely not neurotrophic by themselves. However, they can dramatically increase the potency of select neurotrophins, fibroblast growth factor-2, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF). In the case of GDNF, we have shown that GDNF fails to promote the survival of highly purified neuron populations in vitro unless it is supplemented with TGF-beta. This also applies to the in vivo situation, where antibodies to all three TGF-beta isoforms fully prevent the trophic effect of GDNF on axotomized, target-deprived neurons. In addition to the TGF-beta isoforms -beta2 and -beta3, other members of the TGF-beta superfamily are expressed in the nervous system having important roles in embryonic patterning, cell migration, and neuronal transmitter determination. We have cloned and expressed a novel TGF-beta, named growth/differentiation factor-15 (GDF-15). GDF-15 is synthesized in the choroid plexus and released into the CSF, but also occurs in all regions investigated of the developing and adult brain. GDF-15 is a potent trophic factor for developing and 6-OHDA-lesioned midbrain dopaminergic neurons in vitro and in vivo, matching the potency of GDNF.

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2 −/− hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.

Adrenal chromaffin cells and sympathetic neurons are related, but phenotypically distinct derivatives of the neural crest. Molecular cues that determine the chromaffin cell phenotype have not yet been identified; in contrast to a widely held belief, glucocorticoid signaling is apparently not relevant (Development 126 (1999) 2935). Transforming growth factor-betas (TGF-betas) regulate various aspects of embryonic development and are expressed in the environment of sympathoadrenal (SA) progenitor cells. We have previously shown that neutralization of endogenous TGF-beta from E4 to E8 in the quail embryo significantly increases numbers of adrenal tyrosine hydroxylase-positive cells. Whether endogenous TGF-beta may also be involved in influencing phenotypic development of adrenal chromaffin cells and their SA progenitors has not been analyzed. We now demonstrate that neutralization of endogenous TGF-beta1, -beta2 and -beta3 with a pan-anti-TGF-beta antibody in quail embryos during distinct time windows does not alter phenotypic development of chromaffin cells. In situ hybridizations revealed unaltered expression of neurofilament (NF-160), synaptotagmin I and neurexin I in adrenal glands. Likewise, the NF-associated antigen 3A10, and polyphosphorylated NF epitopes (RT 97) were unaltered. Most importantly, the typical ultrastructure of adrenal chromaffin cells including their large chromaffin secretory granules, a hallmark of the neuroendocrine phenotype, which distinguishes them from sympathetic neurons, was not affected. We therefore conclude that neutralization of endogenous TGF-beta influences chromaffin cell proliferation, but does not interfere with the development of the typical chromaffin cell phenotype.

Fibroblast growth factor 2 (FGF-2) and glial cell line-derived neurotrophic factor (GDNF) support survival of dopaminergic midbrain neurons. Neurons are coupled by gap junctions, propagating metabolites and intracellular second messengers possibly mediating growth factor effects. We asked, therefore, whether gap junctions influence the survival-promoting effects of FGF-2 and GDNF. RT-PCR, Western blotting, and immunocytochemistry demonstrate that FGF-2 but not GDNF upregulates cx43 mRNA and immunoreactivity in rat embryonic day 14 midbrain cultures, whereas cx26, cx32, and cx45 were unchanged. In addition, functional coupling as assayed by the spread of neurobiotin was increased by FGF-2. Furthermore, the gap junction blocker oleamide abolished survival-promoting effects of FGF-2 on dopaminergic midbrain neurons. Together, these results support a direct role of gap junction communication for survival-promoting effects of FGF-2 on dopaminergic midbrain neurons, making gap junction communication a substantial parameter for neuron survival.

The functional anatomy of sympathetic preganglionic neurons is described at molecular, cellular, and system levels. Preganglionic sympathetic neurons located in the intermediolateral column of the spinal cord connect the central nervous system with peripheral sympathetic ganglia and chromaffin cells inside and outside the adrenal gland. Current knowledge is reviewed of the development of these neurons, which share their origin with progenitor cells, giving rise to somatic motoneurons in the ventral horn. Their connectivities, transmitters involved, and growth factor receptors are described. Finally, we review the distribution and functions of trophic molecules that may have relevance for development and maintenance of preganglionic sympathetic neurons.

This review briefly describes the cellular distribution and documented roles of the transforming growth factor (TGF)-beta isoforms TGF-beta2 and -beta3 in the central and peripheral nervous system. TGF-beta2 and -beta3 are coexpressed in developing radial glial and mature astroglial and Schwann cells, as well as in subpopulations of differentiated neurons, most prominently in cortical, hippocampal, and brainstem/spinal cord motor neurons. In vitro studies have suggested a number of potential, physiologically relevant functions for TGF-betas including regulation of astroglial cell proliferation, expression of adhesion molecules, survival promoting roles for neurons in combination with established neurotrophic factors, and differentiative actions on neurons.

We show that following immunoneutralization of endogenous transforming growth factors beta (TGF-beta) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-beta signaling by treatment with a TbetaR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-beta rescued the TGF-beta-deprived phenotype. We conclude that TGF-beta is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.

Serotonin (5-HT) is an important factor controlling survival, differentiation, and plasticity of neurons in serotonergic target regions of the brain and has been implicated in major psychiatric and autonomic disorders. Relatively little is known, however, of factors controlling differentiation and plasticity of developing and adult 5-HT neurons. We show now that 5-HT, the 5-HT1(A) receptor, brain-derived neurotrophic factor (BDNF), and its receptor, trkB, form an auto/paracrine loop for the regulation of the serotonergic phenotype. Serotonin applied to cultures from E14 rat raphe increased numbers of neurons expressing serotonergic markers in a dose-dependent manner. Agonists of the 5-HT1(A) receptor, BP-554 and 8-OH-DPAT, but not agonists of the 5-HT1(B) and 5-HT1(D) receptors, mimicked this effect, while the specific 5-HT1(A) antagonist, WAY-100635, inhibited it. Serotonin also increased BDNF mRNA and protein in embryonic raphe cultures. Induction of serotonergic markers by serotonin was suppressed by a trkB-IgG fusion protein but not by trkC-IgG. Taken together, our data indicate that serotonin acts on 5-HT1(A) autoreceptors, causing up-regulation of BDNF, which activates trkB to promote serotonergic phenotype-specific markers.

Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that fibroblast growth factor (FGF)-2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia 22:19–30, 1998). Other members of the FGF family expressed in the CNS include FGF-5 and FGF-9. We show that both FGF-5 and FGF-9, like FGF-2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF-5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF-9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region-specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT-PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR-2 and FGFR-3. In summary, our results provide evidence for an important role of FGF-2, -5, and –9 in a distinct, CNS region-specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression. GLIA 30:231–241, 2000. © 2000 Wiley-Liss, Inc.

This article summarizes and interprets recent data from our laboratories suggesting that transforming growth factor-ss (TGF-ss1, -ss2, -ss3) is essentially required, in vitro and in vivo, for the neurotrophic signaling of glial cell line-derived neurotrophic factor (GDNF). TGF-ss, which is synthesized by and released from neurons, also synergizes with neurotrophins and members of the neurokine and fibroblast growth factor families by increasing their efficacies. However, when applied to purified neuron populations without other factors being added, TGF-ss does not promote survival or differentiation. Together, these data suggest that neither TGF-ss nor GDNF fulfil essential criteria of a typical neurotrophic factor, as e.g. nerve growth factor (NGF). Moreover, the neurotrophic activity of NGF and other classic neurotrophic factors is apparently based, to a significant extent, on their co-operativity with TGF-ss. Mechanisms, by which TGF-ss generates neurotrophic effects and synergizes with other cytokines are beginning to emerge. Recruitment and/or stabilization of receptors and cross-talks at different levels of signal transduction are likely to be implied in generating the neurotrophic potential of the TGF-ss/cytokine synergisms. Together, these data outline a novel role of TGF-ss in a key event of nervous system development, ontogenetic neuron death. Conceptually more important, however, may be the broadening of the neurotrophic factor concept, which now has to imply the possibility that two cytokines, each being ineffective by itself, become neurotrophically active when acting in concert.

We have cloned, expressed, and raised antibodies against a novel member of the TGF-s superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-s superfamily and does not belong to one of the known TGF-(3 subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.

We have isolated the rat, mouse and human genes of a distant member of the TGF-β superfamily, growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1) by screening of genomic libraries. All three genes are composed of two exons, and contain one single intron that interrupts the coding sequences at identical positions within the prepro-domain of the corresponding proteins. The predicted proteins contain the structural hallmarks of members of the TGF-β superfamily, including the seven conserved carboxy-terminal cysteine residues that form the cystine knot. The orthologous molecules show the lowest sequence conservation of all members of the TGF-β superfamily. RT-PCR reveals an abundant expression of GDF-15/MIC-1 mRNA in numerous embryonic and adult organs and tissues. Promoter analysis of the rat promoter indicates the presence of multiple regulatory elements, including a TATA-like sequence as well as several SP1, AP-1 and AP-2 sites. Deletion analysis suggests that a 350 bp region upstream of the start of the open reading frame appears to be the most important for regulation of transcription.

Transforming growth factor- β s are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor- β s are synthesized, stored and released by the neuroendocrine chromaffin cells, which also express the transforming growth factor- β receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2′-deoxyuridine pulse labeling and tyrosine hydroxylase immunocytochemistry as a marker for young postnatal rat chromaffin cells, we show that treatment with fibroblast growth factor-2 (1 nM) and insulin-like growth factor-II (10 nM) increased the fraction of 5-bromo-2′-deoxyuridine-labeled nuclei from 1% to about 40% of the cells in the absence of serum. In the presence of fibroblast growth factor-2 and insulin-like growth factor-II, transforming growth factor- β 1 (0.08 nM) reduced 5-bromo-2′-deoxyuridine labeling by about 50%, without interfering with chromaffin cell survival or death. Doses lower and higher than 0.08 nM were less effective. Similar effects were seen with transforming growth factor- β 3. In contrast to transforming growth factor- β , ciliary neurotrophic factor, which inhibits proliferation of sympathetic progenitor cells, was not effective on rat chromaffin cells from postnatal day 6. Glucocorticoids also suppress DNA synthesis in fibroblast growth factor-2/insulin-like growth factor-II-treated chromaffin cells. This effect was not mediated by chromaffin cell-derived transforming growth factor- β , as shown by addition of neutralizing antibodies. We conclude that one function of adrenal medullary transforming growth factor- β may be to act as a negative regulator of chromaffin cell division.

We have studied the regulation of survival and serotonergic markers by neurotrophins and several trophically active cytokines in neurons cultured from the embryonic rat raphe region under defined conditions. At embryonic day 14, saturating concentrations of brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 and basic fibroblast growth factor elicited a 2- to 2.5-fold increase in numbers of tryptophan hydroxylase- and serotonin-immunoreactive neurons over a four-day culture period. Transforming growth factor beta-1 and glial cell line-derived neurotrophic factor were less potent, while fibroblast growth factor-5 was only marginally effective. Distinct responses to different factors were noted depending on embryonic age and regional origin of serotonergic neurons. Thus, brain-derived neurotrophic factor augmented numbers of tryptophan hydroxylase-positive neurons at embryonic day 16 by a factor of 7, but only 1.5- to 2-fold when cultures were established from day 13 or 14 embryos. In cultures of rostral serotonergic groups (B4–B9), numbers of tryptophan hydroxylase-positive neurons decreased in the absence of factors, whereas numbers of tryptophan hydroxylase-immunoreactive neurons in cultures from caudal serotonergic groups (B1–B3) increased during a 12-day culture period. There was no evidence that serotonergic neurons undergo apoptosis (as visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling) or proliferate (as visualized by 5-bromodeoxyuridine incorporation) in culture. Numbers of serotonergic neurons also increased when cultures were treated with a brief 24-h pulse of brain-derived neurotrophic factor, supporting the notion that changes in numbers of serotonergic neurons reflected alterations of phenotype rather than cell death or proliferation. The ability of cells to specifically take up the serotonin analog 5,7-dihydroxytryptamine was also up-regulated by brain-derived neurotrophic factor in both rostral and caudal raphe cultures. Lability of the serotonergic phenotype was further suggested by the observation that ciliary neurotrophic factor fully prevented the brain-derived neurotrophic factor-mediated increase in tryptophan hydroxylase-positive neurons. The effect of ciliary neurotrophic factor was dependent on the presence of astrocytes. We conclude that serotonergic neurons show spatially and temporally distinct responses to neurotrophic factors, which seem to have a profound influence of the transmitter phenotype rather than on survival.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7–T10 that project to the adrenal medulla.In situhybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked α receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-β (TGF-β) receptor II. Administration of GDNF (recombinant human, 1 μg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochromecapplied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-β isoforms but not GDNF. This suggests that the presence of endogenous TGF-β was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-β for permitting trophic actions of GDNFin vitro(Krieglstein et al., 1998) also applies to thein vivosituation.

Fibroblast growth factor (FGF)-2 is expressed by astroglial cells and regulates astroglial proliferation, differentiation, and gap junction communication. We show now that uncoupling of astroglial cells cultured from neonatal rat cortex, striatum and mesencephalon by 18alpha-glycirrhetinic acid (AGA) or arachidonic acid (AA) (demonstrated by intracellular injections of Lucifer Yellow) does affect neither levels of FGF-2 mRNA (investigated by a competitive RT-PCR method), nor protein (as revealed by Western blot analysis). This suggests that expression of FGF-2, which reduces gap junction communication of cortical and striatal astroglial cells [15], is not affected when astroglial cells are uncoupled. Regulation of FGF-2 expression and coupling in astroglial cells are thus not reciprocal suggesting more complex interrelationships between these two important parameters of astroglial cell differentiation.

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7–T10, disappear. Administration of NT-4 in gelfoams (6 μg) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7–T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT-PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non-neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target-derived factor, as suggested for GDNF, the motoneuron-muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target-derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin.

The aim of this study was to investigate putative effects of calcitonin gene-related peptide on developing dopaminergic neurons in the ventral mesencephalon. To determine a time-point for a physiological role of calcitonin gene-related peptide in the development of this system, we first investigated calcitonin gene-related peptide messenger RNA expression in the ventral mesencephalon of Wistar rats at embryonic days (E) 11–19. Calcitonin gene-related peptide messenger RNA was not detectable at E11, i.e. prior to the appearance of dopaminergic neurons in this area. From E14 to E19, calcitonin gene-related peptide messenger RNA was expressed in increasing amounts. We therefore investigated the effects of calcitonin gene-related peptide on serum-free cell cultures established from the E14 midbrain floor. Addition of calcitonin gene-related peptide (200 ng/ml) every other day significantly increased neuronal differentiation, including longer tyrosine hydroxylase-positive neurites, enhanced immunoreactivity for growth-associated protein-43 and increased dopaminergic uptake per neuron. These effects were maximal after seven to eight days. Calcitonin gene-related peptide acted synergistically with fibroblast growth factor-2 on these parameters. In contrast to fibroblast growth factor-2, however, calcitonin gene-related peptide did not promote survival of tyrosine hydroxylase-immunoreactive neurons. Lack of calcitonin gene-related peptide expression in the mesencephalon at E11 was paralleled by a lack of effect of calcitonin gene-related peptide on early presumptive dopaminergic neurons in terms of eliciting this phenotype. Our data suggest that calcitonin gene-related peptide may act physiologically as a differentiation-promoting factor for phenotypically defined dopaminergic neurons during a time period when dopaminergic neurons assemble in the ventral mesencephalon and grow axons towards their targets.

Growth/differentiation factor-5 (GDF-5), a morphogenetic protein, has previously been shown to act as a neurotrophic factor for midbrain dopaminergic neurons. To further elucidate the neurotrophic potential of GDF-5, serum free cultures of dorsal root ganglionic (DRG) neurons from developing chick embryos were treated with GDF-5 with or without the simultaneous addition of other trophic factors. Our results show that GDF-5 has a minor promoting effect on its own, but it can enhance the survival promoting effect of neurotrophin-3 (NT-3) and nerve growth factor (NGF) on cultured DRG neurons. Our finding fits well into the concept that neurotrophic factors may act synergistically in ensuring survival of different neuronal populations. The capacity of GDF-5 to reduce the requirement of a subpopulation of sensory neurons for NT-3 may have implications for the treatment of peripheral neuropathies.

Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.

TrkA high-affinity receptors are essential for the normal development of sympathetic paravertebral neurons and subpopulations of sensory neurons. Paravertebral sympathetic neurons and chromaffin cells of the adrenal medulla share an ontogenetic origin, responsiveness to NGF, and expression of TrkA. Which aspects of development of the adrenal medulla might be regulated via TrkA are unknown. In the present study we demonstrate that mice deficient for TrkA, but not the neurotrophin receptor TrkB, show an early postnatal progressive reduction of acetylcholinesterase (AChE) enzymatic activity in the adrenal medulla and in preganglionic sympathetic neurons within the thoracic spinal cord, which are also significantly reduced in number. Quantitative determinations of specific AChE activity revealed a massive decrease (−62%) in the adrenal gland and a lesser, but still pronounced, reduction in the thoracic spinal cord (−40%). Other markers of the adrenal medulla and its innervation, including various neuropeptides, chromogranin B, secretogranin II, amine transporters, the catecholamine-synthesizing enzymes tyrosine hydroxylase and PNMT, synaptophysin, and L1, essentially were unchanged. Interestingly, AChE immunoreactivity appeared unaltered, too. Preganglionic sympathetic neurons, in contrast to adrenal medullary cells, do not express TrkA. They must, therefore, be affected indirectly by the TrkA knock-out, possibly via a retrograde signal from chromaffin cells. Our results suggest that signaling via TrkA, but not TrkB, may be involved in the postnatal regulation of AChE activity in the adrenal medulla and its preganglionic nerves.

We present the nucleotide (nt) sequence for the cDNA encoding bovine brain-derived neurotrophic factor (BDNF). The nt and peptide sequences were compared to those of other BDNF genes from other species.

Publisher Summary In this chapter several studies have been summarized to reveal the pattern of expression of several candidate markers in the embryonic adrenal gland of wild-type mice. It has been suggested that Phox 2 is a marker for both differentiated and presumptive catecholaminergic adrenal chromaffin cells prior to reaching detectable levels of tyrosine hydroxylase (TH) immunoreactivity. In addition to TH, Phox 2, peanut agglutinin (PNA) and neuropeptide Y (NPY) are detectable in all postnatal chromaffin cells. Noradrenergic chromaffin cells possess the full repertoire of chromaffin cell functions except the capacity to synthesize adrenaline. Adult noradrenergic chromaffin cells exhibit at least three markers that they do not share with adrenergic chromaffin cells, GAP43, the adhesion molecule L1, and vesicular monoamine transporter-2 (VMAT2). The data presented in the chapter provide a useful basis for further analyzing the phenotypes of adrenal chromaffin cells in glucocorticoid-receptor knockout mice. Furthermore, the results suggest that one marker found predominantly or exclusively in mature adrenergic chromaffin cells in mice, chromogranin B can be detected in chromaffin progenitor cells prior to the expression of other key markers for the adrenergic phenotype, phenylethano1amine-Nmethyltransferase (PNMT), and secretogranin II. This suggests that differentiative properties of adrenergic chromaffin cells can be regulated sequentially and are probably, in part, independent from glucocorticoid signaling.

Glial cell line-derived neurotrophic factor (GDNF) is a widely distributed member of the transforming growth factor-beta superfamily and a potent neurotrophic molecule for several neuron populations in the peripheral and central nervous system. We show here that adrenal medullary chromaffin cells synthesize GDNF mRNA and contain immunoreactive GDNF protein. GDNF immunoreactivity can be found as early as embryonic day 16 in chromaffin progenitor cells of the rat adrenal gland and becomes more prominent with age. Most of the chromaffin cells within the adult rat adrenal medulla are GDNF immunoreactive, including both the noradrenergic and adrenergic subpopulations. The functions of adrenal medullary GDNF are still enigmatic but may include both auto/paracrine roles and retrograde trophic support of preganglionic neurons in the spinal cord or of sensory neurons that innervate chromaffin cells.

Neurotrophins are established survival and differentiation factors for sensory dorsal root ganglionic (DRG) neurons. We have previously shown that proteins from the secretory granules of adrenal chromaffin cells have a capacity to promote the survival of cultured chick DRG neurons. Using DRG neurons from embryonic day (E) 8 chick embryos we show now that this material is (i) as effective as nerve growth factor (NGF), (ii) additive to NGF, neurotrophin-3, or -4, (iii) unlikely to be a neurotrophin, since the survival promoting effect can not be blocked by K252b, a specific inhibitor of the signal transduction pathways of neurotrophin high affinity receptors, (iv) partially blockable by antibodies to transforming growth factor-beta (TGF-beta) 1/2/3, and (v) more potent than any other out of 30 cytokines tested individually, including fibroblast growth factor (FGF)-5, epidermal growth factor (EGF), TGF-alpha, platelet-derived growth factor (PDGF)-AB, insulin-like growth factors (IGF)-I and -II, leukemia inhibitory factor (LIF), TGF-beta, glial cell line-derived neurotrophic factor (GDNF), stem cell factor, granulocyte-colony stimulating factor (G-CSF), oncostatin M, tumor necrosis factor (TNF)-alpha, and interleukins (IL)-1 through -12. We conclude that chromaffin cells, which are known to receive a sensory innervation, can provide (a) trophic factor(s), which, in addition to neurotrophins, may be relevant for the maintenance of DRG neurons.

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk -signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.

Chromaffin cells, the neuroendocrine cells of the adrenal medulla and paraganglia, occupy paradigmatic roles in molecular, cellular and developmental neurobiology. The fact that they are very effective 'minipumps', secreting numerous bioactive substances including amines, neuropeptides and proteins, has made them interesting and useful for the treatment of chronic pain and Parkinsonism. An essential advantage of chromaffin cells is that they can be isolated and purified in extremely high numbers, which can never be achieved for peripheral or central nervous system neurons. Growth factors (cytokines) and peptides with growth factor-like efficacies constitute an important component of bioactive materials released from chromaffin cells. Not only their presence, but also neural and humoral mechanisms regulating their expression and release, are now being revealed. Prominent examples include fibroblast growth factors (FGFs), transforming growth factor-beta s (TGF-beta s) and interleukins. Functions that can be assigned to these factors in the adrenal medulla are gradually emerging. For example, FGFs and TGF-beta s can regulate chromaffin cell proliferation and differentiation and participate in the neurotrophic maintenance of neurons innervating chromaffin cells. In contrast, the functions of the predominant secretory proteins of chromaffin cells, the chromogranins, are still largely unknown, but might include cytokine-like roles. Thus, chromaffin cells continue to teach neurobiologists about the fundamental capacity of neurons to secrete bioactive molecules with a wide range of functions as well as modes of their secretion underscoring the close relationship of endocrine and neuronal systems.

Fibroblast growth factor-2 (FGF-2), locally administered in gelfoam to the striatum of mice treated with the neurotoxic drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has restorative and neuroprotective effects on dopaminergic neurons and associated striatal transmitter systems. Most of the beneficial alterations are apparently indirect. FGF-2 must therefore act through a series of cellular and molecular intermediate steps, which have not been explored. We have previously shown that FGF-2 does not significantly affect the astroglial reaction at the time, when the neuroprotective effect of FGF-2 reaches a peak (Day 11). In this study we have investigated the effect of FGF-2 at earlier time points after MPTP treatment. We report now that as early as 6 h after administration of the gelfoam containing either FGF-2 or control protein, FGF-2 immunoreactivity disappears from astroglial nuclei, while appearing in small ramified GFAP- and S-100-negative cells, most likely microglia. At 18 h, numbers and staining intensities of GFAP-ir astroglial cells are greater in FGF-2- than in cytochrome C-treated animals. At this time FGF-2-ir reappears in astroglia nuclei of cytochrome C-treated animals, but remains undetectable in the striatum carrying the FGF-2-containing gelfoam. Ramified GFAP/S-100-negative presumed microglial cells are now intensely ir for FGF-2. Signs of an FGF-2-mediated astrogliotic reaction are very pronounced at 18 h and 2 days, but no longer at 11 days, when the astrogliosis reaction has become equally strong in FGF-2- and cytochrome C-treated striata. Our results suggest that administration of FGF-2 to the MPTP-lesioned striatum has early effects on astro- and presumed microglia cells, notably on the nuclear FGF-2-ir of astrocytes. These changes may be involved in mediating the neuroprotective effects of FGF-2 in the MPTP-model of Parkinsonism.

Fibroblast growth factors (FGFs) can have pronounced effects on cells of the nervous system. The expression of FGFs at both the RNA and protein level is generally very low in normal adult tissues. Regulation of the expression of FGF genes is apparently necessary, as FGFs have transforming potential, and overexpression of these growth factors is frequently associated with a transformed phenotype. All members of the FGF family with the exception of FGF-1 and -2 possess consensus sequences for addition of sugar residues and apparently may be glycosylated. The FGF receptor (FGFR) types are most similar in the tyrosine kinase domain, a region that is also homologous to the tyrosine kinase domain of other growth factor receptors. The immunoglobulin loops 2 and 3 are moderately conserved while the immunoglobulin loop 1 is most divergent between receptor types. The results describing the distribution of FGF-1 and FGF-2 proteins and their mRNAs in the central nervous system are found to be consistent in some parts, but inconsistent in others.

The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), are established survival promoting molecules for dopaminergic (DAergic) neurons cultured from the fetal rat midbrain floor. We have cultured and compared the survival of embryonic day (E) 14 mesencephalic cells in fully defined, serum-free medium, with serum-primed cultures (one hour during dissociation). Cultures were characterized using antibodies against neuron-specific enolase (NSE), tyrosine hydroxylase (TH), vimentin, glial fibrillary acidic protein (GFAP), and the antigen A2B5. The absolute absence of serum did not reduce the survival of TH-positive DAergic neurons nor alter the percentages of cells staining for the above markers. Transforming growth factor-β3 (TGF-β3) and glial cell line-derived neurotrophic factor (GDNF), two members of the TGF-β superfamily, both promoted the survival of TH-positive cells (TGF-β3: 2-fold; GDNF: 1.6-fold) over the 8-day culture period. Survival mediated by TGF-β3 and GDNF was independent of whether or not the cells had been initially exposed to serum. In contrast, the survival promoting effects of BDNF and NT-4 were crucially dependent on serum priming. RT-PCR for the full-length trkB high affinity neurotrophin receptor revealed its presence in both culture systems. We conclude that priming with serum is important to make DAergic neurons fully responsive to BDNF and NT-4. Underlying mechanisms might be sought at the level or distal of trkB receptor expression, without excluding the possiblity that serum elicits production of growth factors that synergistically act with neurotrophins in these cultures.

The heparin-binding, growth-associated molecule (HB-GAM) is a developmentally regulated protein that belongs to a new family of heparin-binding molecules, not related to the fibroblast growth factors (FGFs), with putative functions during cell growth and differentiation. In order to further study the functional role of HB-GAM we have used a polyclonal antiserum, raised against the purified protein to localize HB-GAM in the developing and adult rat cerebellar cortex. During postnatal development HB-GAM-like immunoreactivity (IR) was found to be present in all layers of the cerebellar cortex. IR was mainly associated with processes or extracellular structures but not with cell bodies. Throughout all the stages examined the molecular layer was clearly labeled, whereas staining in the internal granular layer was diffuse. IR in the external granular layer on postnatal day 1 and 8 was found to be associated with radially oriented fibres connecting the internal granular layer with the pial surface of the cerebellum. The intensity of this staining seemed to increase from day 1 to 8. Staining of corresponding areas with an antiserum against the glial fibrillary acidic protein (GFAP) suggested that the HB-GAM antiserum in the developing cerebellar cortex labers Bergmann glia fibres of Golgi epithelial cells. Because of the diffuse staining of the molecular layer in the adult rat it was not possible to distinguish whether radial fibres in the adult contained any HB-GAM IR. Golgi epithelial cells are considered as crucial for the migration of granular cells during the differentiation of the cerebellar cortex. We therefore speculate that the association of HB-GAM-like IR may be of functional relevance. The fact that molecules, such as tenascin, known to be involved in morphogenetic events show a similar spatial temporal distribution pattern further underscores this hypothesis. HB-GAM, which possesses a classical signal sequence, might be released in the extracellular space and could mediate adhesion phenomena by binding to heparin-like molecules associated with the neuronal membrane. Therefore, it will be important to investigate whether specific antibodies against HB-GAM are able to interfere with normal cerebellar development in vitro and in vivo.

The synthesis of the neuropeptide galanin (GAL) is greatly enhanced after axonal lesion in different neuron populations of the peripheral and central nervous system. In sympathetic ganglia, GAL-immunoreactive nerve fiber baskets have been found surrounding postganglionic neurons after axotomy. Until now, it is unclear if GAL may be involved in neuronal survival or regeneration as suggested for vasoactive intestinal peptide (VIP) that is also upregulated after nerve lesion. We have, therefore, studied the effects of GAL on survival of sympathetic neurons dissociated from newborn rat superior cervical ganglia and on neurite outgrowth of PC12 cells. These effects were compared to those elicited by VIP. Whereas VIP promoted survival of about 10% of sympathetic neurons 2 days after nerve growth factor deprivation and induced neurite outgrowth of PC12 cells already at 6 h after addition of the peptide, GAL had no effect in either of these culture systems. While the induction of VIP may be beneficial for axotomized neurons, the functional significance of increased GAL levels remains to be established.