Differential expression of FGF-2 isoforms in the rat adrenal medulla during postnatal development in vivo

Basic fibroblast growth factor (FGF-2) isoforms of the adrenal medulla are differentially expressed during rat postnatal development. While the 18 and 23 kDa isoforms continuously rise towards the adult expression level, the 21 kDa isoform displays a peak expression at postnatal day 28. The peak expression of the 21 kDa isoform correlates with the peak of the corticosterone concentration during postnatal development. Together with the previously demonstrated increase of the 21 kDa isoform in the adrenal medulla in vivo after glucocorticoid administration these results suggest that the differential regulation of the FGF-2 isoforms could be a physiologically occurring mechanism.     

Fibroblast growth factor-2 deficiency causes defects in adult hippocampal neurogenesis, which are not rescued by exogenous fibroblast growth factor-2

Neurogenesis within the adult brain is restricted to selected areas, one of which is the dentate gyrus (DG). Several growth factors have been reported to affect neurogenesis in the adult DG. However, a role of fibroblast growth factor-2 (FGF-2) in adult hippocampal neurogenesis has not been firmly established. We have analyzed neurogenesis in the DG using in vivo and in vitro approaches. FGF-2(-/-) mice revealed no alterations in the number of proliferating cells but a significant decrease in the numbers of newly generated neurons. Moreover, FGF-2 added to hippocampal slice cultures from FGF-2(-/-) mice was unable to rescue the phenotype. Although an increase in death of neurogenic cells in the FGF-2-deficient DG could not be specifically demonstrated, there was a massive increase in global cell death in FGF-2(-/-) hippocampal slice cultures compared with slices from wild-type mice. Cell death could not be prevented by addition of FGF-2. Neutralization of endogenous FGF-2 in hippocampal slices did not interfere with neurogenesis in a short-term paradigm. Together, our data suggest that FGF-2 is essentially required for maturation of new neurons in adult hippocampal neurogenesis but is likely to operate synergistically in combination with other mechanisms/growth factors.     

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.     

FGF-18 is a neuron-derived glial cell growth factor expressed in the rat brain during early postnatal development

We examined the expression of fibroblast growth factor-18 (FGF-18) in the rat brain during postnatal development by in situ hybridization. FGF-18 was transiently expressed at the early postnatal stages in various regions of the rat brain including the cerebral cortex and hippocampus. FGF-18 in the brain was preferentially expressed in neurons but not in glial cells. To elucidate the role of FGF-18 in the brain, we examined the ligand-specificity of FGF-18 by the BIAcore system. FGF-18 was found to bind to FGF receptors (FGFRs)-3c and -2c but not to FGFR-1c, suggesting that FGF-18 acts on glial cells but not on neurons. Therefore, we examined the mitogenic activity of FGF-18 for cultured rat astrocytes and microglia. FGF-18 was found to have mitogenic activity for both astrocytes and microglia. We also examined the neurotrophic activity of FGF-18 for cultured rat cortical neurons. FGF-18 was found to have no neurotrophic activity. The present findings indicated that FGF-18 is a unique FGF that plays a role as a neuron-derived glial cell growth factor in early postnatal development when gliogenesis occurs.     

Expression of plasma membrane Ca2+ ATPase family members and associated synaptic proteins in acute and cultured organotypic hippocampal slices from rat

Plasma membrane Ca2+ ATPases (PMCAs) are critical regulators of intracellular Ca2+ concentration ([Ca2+]i). Specific isoforms have also been demonstrated to interact and co-localise with members of the synapse-associated protein (SAP) family in hippocampal dendritic spines. Presently, only indirect evidence of changes in PMCA protein expression during postnatal development exists, therefore we chose to examine the postnatal developmental protein expression patterns of PMCAs 1-4 and the SAP proteins SAP102 and PSD95. Using Western blotting analysis, we compared the postnatal expression in the in vivo hippocampus to the expression within in vitro organotypic hippocampal slice cultures; a valid model of the developing hippocampus. All PMCA and SAP family members studied showed a marked increase in protein expression levels throughout the postnatal time course both in vivo and in vitro. SAP102 and the ubiquitously expressed PMCAs 1 and 4 followed a similar time course of expression within the in vivo and in vitro preparations. In contrast, the neurone-specific PMCA isoforms 2 and 3 and PSD95 displayed slight differences in early postnatal development. However, and most importantly, their expression > or = 14 days in vitro (DIV) was similar to that in vivo.The results of this study demonstrate that postnatal expression of all PMCAs, SAP102 and PSD95 is similar in both the in vivo hippocampus and the in vitro organotypic hippocampal slice culture. Our results support the use of organotypic hippocampal slice cultures for future investigations of the importance of PMCAs for neuronal Ca2+ handling and SAP family member interactions.     

Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus

Insulin-like growth factor-1 (IGF-1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF-1 and -2) signaling is mediated primarily by the type 1 IGF receptor (IGF-1R) and modulated by six high-affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF-2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP-1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF-1, IGFBP-2, -3, and -5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF-2, IGF-2R, and IGFBP-6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF-1R and IGFBP-4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF-1, IGF-1R, IGF-2R, IGFBP-2, and -4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF-1 and IGF-1R (by 130% and 86%, respectively), lower levels of IGF-2R (by 72%), and similar levels of IGFBP-4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.     

Myelin-associated glycoprotein inhibits neurite/axon growth and causes growth cone collapse

We have previously shown that myelin-associated glycoprotein (MAG) inhibits neurite growth from a neuronal cell line. In this study we show that 60% of axonal growth cones of postnatal day 1 hippocampal neurons collapsed when they encountered polystyrene beads coated with recombinant MAG (rMAG). Such collapse was not observed with denatured rMAG. Neurite growth from rat embryonic hippocampal and neonatal cerebellar neurons was also inhibited about 80% on tissue culture substrates coated with rMAG. To investigate further the inhibitory activity of MAG in myelin, we purified myelin from MAG-deficient mice and separated octylglucoside extracts of myelin by diethylaminoethyl (DEAE) ion-exchange chromatography. Although there was no significant difference in neurite growth on myelin purified from MAG-/- and MAG+/+ mice, differences were observed in the fractionated material. The major inhibitory peak that is associated with MAG in normal mice was significantly reduced in MAG-deficient mice. These results suggest that although MAG contributes significantly to axon growth inhibition associated with myelin, its lack in MAG-deficient mice is masked by other non-MAG inhibitors. Axon regeneration in these mice was also examined after thoracic lesions of the corticospinal tracts. A very small number of anterogradely labeled axons extended up to 13.2 mm past the lesion in MAG-/- mice. Although there is some enhancement of axon generation, the poor growth after spinal cord injury in MAG-/- mice may be due to the presence of other non-MAG inhibitors. The in vitro studies, however, provide the first evidence that MAG modulates growth cone behavior and inhibits neurite growth by causing growth cone collapse. © 1996 Wiley-Liss, Inc.     

Distribution of fibroblast growth factor 5 mRNA in the rat brain: an in situ hybridization study

Fibroblast growth factors (FGFs) are potent growth factors with roles in the brain ranging from development to adult plasticity. FGF-5 is a newly described member of the fibroblast growth factor family. In order to evaluate a possible role of FGF-5, we have examined the locus of synthesis of FGF-5 in the rat brain. We have used in situ hybridization of³⁵S-labelled RNA probe complementary to FGF-5 mRNA. FGF-5 mRNA was present in neurons in select regions of the rat brain. FGF-5 mRNA expression was particularly intense in the olfactory bulb within periglomerular elements and the mitral cell layer. The primary olfactory cortex also showed a robust expression of FGF-5 mRNA mostly within layer II. In the hippocampal formation, the greatest labelling of FGF-5 mRNA occurred in hippocampal pyramidal cells within subfields CA3 and secondarily within subfields CA1, CA2 and CA4. The dentate gyrus granule cells displayed a modest hybridization signal. The cerebral cortex (neocortex) showed a light labelling throughout its rostro-caudal extent mostly within external layers. The entorhinal cortex showed a higher FGF-5 mRNA expression as compared to the neocortex and signal appeared more intense in layer II. In general, FGF-5 mRNA was shown to be localized mostly in limbic structures, suggesting that FGF-5 may play a role in limbic system function of dysfunction.     

The physiological and pharmacological role of basic fibroblast growth factor in the dopaminergic nigrostriatal system

Basic fibroblast growth factor (FGF-2) is a physiological relevant neurotrophic factor in the nigrostriatal system and hence a promising candidate for the establishment of alternative therapeutic strategies in Parkinson's disease. FGF-2 and its high-affinity receptors (FGFR) display an expression in the developing, postnatal, and adult substantia nigra (SN) and in the striatum. Exogenous application promoted survival, neurite outgrowth and protection from neurotoxin-induced death of dopaminergic (DA) neurons both in vitro and in vivo. In animal models of Parkinson's disease, co-transplantation of fetal DA cells with FGF-2 expressing cells increased survival and functional integration of the grafted DA neurons resulting in improved behavioral performance. Analyzing the physiological function of the endogenous FGF-2 system during development and after neurotoxin-induced lesion revealed for the DA neurons of the SNpc a dependence on FGFR3 signaling during development. In addition, in the absence of FGF-2 an increased number of DA neurons was found, whereas enhanced levels of FGF-2 resulted in a reduced DA cell density. Following neurotoxin-induced lesion of DA neurons, FGF-2-deleted mice displayed a higher extent of DA neuron death whereas in FGF-2 overexpressing mice more DA neurons were protected. According to the data, FGF-2 seems to promote DA neuron survival via FGFR3 during development, whereas absence of this ligand could be compensated by other members of the FGF family. In contrast, in the adult organism, FGF-2 cannot be compensated by other factors under lesion conditions suggesting a central role for this molecule in the nigrostriatal system.     

Fibroblast Growth Factor-5 Promotes Differentiation of Cultured Rat Septal Cholinergic and Raphe Serotonergic Neurons: Comparison with the Effects of Neurotrophins

Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which has a signal sequence characteristic of secretory proteins. FGF-5 mRNA has previously been shown to be present in the adult mouse brain. Here we demonstrate that recombinant FGF-5 has neurotrophic activity on cultured rat septal cholinergic and raphe serotonergic neurons. The effect of FGF-5 on serotonin uptake was stronger than that evoked with either brain-derived neurotrophic factor or neurotrophin-3. FGF-5 also increased the choline acetyltransferase activity of cultured rat septal cholinergic neurons, the effect being additive to that of nerve growth factor. In situ hybridization experiments and immunohistochemistry using a specific anti-FGF-5 antibody demonstrated that FGF-5 is expressed in rat hippocampal neurons. Like nerve growth factor mRNA, the levels of FGF-5 mRNA in the rat hippocampus increased substantially during early postnatal development. In addition, injection of the muscarinic receptor agonist pilocarpine elevated FGF-5 mRNA. The presence of the secretory FGF-5 in the rat hippocampus, a target field of septal cholinergic and raphe serotonergic neurons, suggests that FGF-5 acts as a trophic factor for these neurons also in vivo.     

Fibroblast growth factor-2 increases functional excitatory synapses on hippocampal neurons

The effect of fibroblast growth factor-2 (FGF-2) on synapse formation was investigated using rat cultured hippocampal neurons. Treatment with FGF-2 (0.4-10 ng/mL) for 6 days enhanced synaptogenesis on these neurons by approximately 50%, as determined by counting puncta immunostained for presynaptic- or postsynaptic-specific proteins. This enhancement was statistically significant, and was abolished by a specific inhibitor of mitogen-activated protein kinase (MAPK). The majority of neurons expressed FGF receptors (types 1-3) abundantly on the membrane of somata, dendrites, and growth cones, and in these regions phosphorylation of MAPK was enhanced after FGF-2 application. Furthermore, our experiments showed that the majority of synapses formed in cultures containing FGF-2 were positive both for presynaptic proteins and postsynaptic excitatory synapse-specific proteins, and that these synapses had a similar capacity to recycle the fluorescent styryl dye FM4-64 as those in the control culture. These results indicate that: (i) FGF-2 increases excitatory synapses on hippocampal neurons by activating MAPK activity through FGF receptors; and (ii) synapses formed in FGF-2-treated culture are capable of cycling vesicles.     

Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats

Although numerous studies have examined the neurotoxicity of acrylamide in adult animals,the effects on neuronal development in the embryonic and lactational periods are largely unknown.Thus,we examined the toxicity of acrylamide on neuronal development in the hippocampus of fetal rats during pregnancy.Sprague-Dawley rats were mated with male rats at a 1:1 ratio.Rats were administered 0,5,10 or 20 mg/kg acrylamide intragastrically from embryonic days 6–21.The gait scores were examined in pregnant rats in each group to analyze maternal toxicity.Eight weaning rats from each group were also euthanized on postnatal day 21 for follow-up studies.Nissl staining was used to observe histological change in the hippocampus.Immunohistochemistry was conducted to observe the condition of neurites,including dendrites and axons.Western blot assay was used to measure the expression levels of the specific nerve axon membrane protein,growth associated protein 43,and the presynaptic vesicle membrane specific protein,synaptophysin.The gait scores of gravid rats significantly increased,suggesting that acrylamide induced maternal motor dysfunction.The number of neurons,as well as expression of growth associated protein 43 and synaptophysin,was reduced with increasing acrylamide dose in postnatal day 21 weaning rats.These data suggest that acrylamide exerts dose-dependent toxic effects on the growth and development of hippocampal neurons of weaning rats.     

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.     

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.     

Hippocampal FGF-2 and FGFR1 mRNA expression in major depression, schizophrenia and bipolar disorder

INTRODUCTION: FGF-2 is important for stem cell proliferation, neocortical development and adult neuronal survival and growth. Reduced frontal cortical FGF-2 expression is described in major depression and is attenuated by antidepressants. We determined the distribution of hippocampal FGF-2 and its receptor (FGFR1) mRNA in post-mortem brains of people who suffered from major depression, bipolar disorder and schizophrenia and those of controls. METHODS: FGF-2 and FGFR1 mRNA were measured within hippocampal CA1, CA4 regions and the dentate gyrus (DG), using in situ hybridization. Within hippocampal regions, cellular staining was compared between diagnostic groups, using repeated measures analysis of variance. RESULTS: The density of FGF-2 mRNA+ cells in CA4 was reduced in depression compared to controls. The percentage of FGFR1 mRNA+ cells was higher in depression (CA1 and CA4) and schizophrenia (CA4) than in controls. FGFR1 mRNA expression was higher in depression than in the other groups in CA1, CA4 and DG. Overall FGF-2 mRNA expression was higher in DG than in CA1 and CA4. CONCLUSIONS: We found raised measures of FGFR1 mRNA+ in major depression and, less so, in schizophrenia, along with reduced FGF-2 mRNA density in depression. Perturbations of FGF regulation could be relevant to the pathogenesis of both disorders as FGF-2 and FGFR1 are implicated in normal hippocampal synaptology, stem cell recruitment, and connectivity, and are modulated by corticosteroids.