specificity of the second messenger pathways involved in basic...

Specificity of the Second MessengerPathways Involved in Basic FibroblastGrowth Factor-Induced Survival andNeurite Growth in Chick CiliaryGanglion Neurons

Alessandra Gilardino,1,2 Silvia Farcito,1,2 Pollyanna Zamburlin,1,2

Chiara Audisio,1 and Davide Lovisolo1,2,3*1Department of Animal and Human Biology, University of Torino, Torino, Italy2Centre of Excellence on Nanostructured Surfaces and Interfaces, University of Torino, Torino, Italy3Neuroscience Institute of Torino, University of Torino, Torino, Italy

Basic fibroblast growth factor (bFGF) exerts multipleneurotrophic actions on cultured neurons from the cili-ary ganglion of chick embryo, among them promotionof neuronal survival and of neurite outgrowth. To under-stand the specificity of the signal transduction cas-cades involved in the control of these processes, weused pharmacological inhibitors of the three main effec-tors known to act downstream of the bFGF receptor(FGFR): phospholipase Cg (PLCg), mitogen-activatedprotein kinase (MAPK), and phosphatidylinositol 3-ki-nase (PI3-K). Neuronal survival was assessed at 24 and48 hr; neurite growth was analyzed both on dissociatedneurons and on explants of whole ganglia. Our datashow that only the PI3-K pathway is involved in the sur-vival-promoting effect of bFGF; on the other hand, allthree effectors converge on the enhancement of neuriteoutgrowth, both on isolated neurons and in whole gan-glia. VVC 2009 Wiley-Liss, Inc.

Key words: fibroblast growth factor-2 (FGF-2); neuriteoutgrowth; neuronal survival; signal transductionpathways; ciliary ganglion neurons

Basic fibroblast growth factor (bFGF, or FGF-2) isa potent neurotrophic factor for many neuronal popula-tions (Walicke et al., 1986; Ferrari et al., 1989; Grotheet al., 1991; Hossain and Morest, 2000; Abe and Saito,2001), acting at different developmental stages (see, e.g.,Haynes, 1988; Westermann et al., 1990; Temple andQian, 1995) and also on adult neurons, where it canexert neuroprotective effects and support regenerationafter injury (Grothe et al., 2006; Grothe and Timmer,2007). Most of the effects on neuronal targets are de-pendent on the binding of the factor to the receptorsubtype FGFR1, the most widely expressed of the fourFGFRs (see, e.g., Ford-Perriss et al., 2001). In chick cil-iary ganglion (CG) neurons, a consolidated model fordevelopmental studies (Dryer, 1994), its effects have

been described in detail, and it has been shown to mod-ulate a broad set of processes, such as neuronal survival(Unsicker et al., 1987, 1992; Dreyer et al., 1989; Distasiet al., 1998), neurite outgrowth (Zamburlin et al., 2006),and proliferation of neuronal precursors (Gilardino et al.,2000).

The spectrum of functions exerted by this factor inthe same experimental model raises the question of thepotentially differential roles of the signal transductionpathways activated downstream of its receptor(s). Thethree most extensively investigated pathways are thoseinvolving the mitogen-activated protein kinase (MAPK),phospholipase Cg (PLCg), and phosphatidylinositol 3-kinase (PI3-K) enzymes (Klint and Claesson-Welsh,1999; Dailey et al., 2005; Eswarakumar et al., 2005).

MAPK (also called ‘‘extracellular signal-regulatedkinase’’; ERK) has been shown to be involved in bFGF-induced neuronal differentiation and neurite growth inembryonic rat hippocampal neurons (Abe et al., 2001);Perron and Bixby (1999), with embryonic chick retinalneurons, have provided evidence for the role of thisenzyme as a point of convergence of different pathwayscontrolling neurite outgrowth. In addition, MAPK acti-vation downstream of the FGFR has been shown to beinvolved in survival of different types of neurons in cul-ture (GT1-7 cell line, Tsai et al., 1995; embryonic rat

The first two authors contributed equally to this work.

Contract grant sponsor: Compagnia di San Paolo Foundation (to the

Centre of Excellence on Nanostructured Surfaces and Interfaces).

*Correspondence to: Davide Lovisolo, Department of Animal and

Human Biology, University of Torino, Via Accademia Albertina 13,

10123 Torino, Italy. E-mail: [email protected]

Received 20 August 2008; Revised 16 March 2009; Accepted 17 March

2009

Published online 29 April 2009 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.22116

Journal of Neuroscience Research 87:2951–2962 (2009)

' 2009 Wiley-Liss, Inc.

cortical neurons, Abe and Saito, 2000; embryonic chickretinal neurons, Desire et al., 2000; embryonic and post-natal rat mesencephalic, hippocampal, and cerebellarneurons, Neiiendam et al., 2004).

The PLCg pathway and its effectors have beenreported to modulate neurite growth in several experi-mental models following FGFR activation (rat cerebellarneurons, Williams et al., 1994; Doherty and Walsh,1996; Hall et al., 1996; Xenopus embryonic retinal neu-rons, Lom et al., 1998; H19-7 cell line, Oh et al.,2007), but limited evidence can be found about theinvolvement of this enzyme in bFGF-promoted neuronalsurvival (Katsuki et al., 2000, on embryonic rat hippo-campal neurons). In embryonic Xenopus retinal axons,Webber et al. (2005) have shown that both of theabove-mentioned pathways are required for bFGF-induced neurite extension, but only the latter is involvedin growth cone orientation.

On the other hand, the role of PI3-K in this con-text is more controversial. According to Klint and Claes-son-Welsh (1999), this enzyme does not play a majorrole in bFGF-induced neuronal differentiation. Webberet al. (2005) reported that the block of PI3-K did notinfluence neurite extension from embryonic Xenopus ret-inal axons promoted by bFGF in vitro; similar findingswere described for postnatal rat cerebellar neurons byWilliams and Doherty (1999). However, Rodgers andTheibert (2002) have provided evidence that this secondmessenger pathway is necessary for sustaining outgrowthand maintenance of neurites. Although no data are avail-able on the direct involvement of this enzyme in bFGF-induced survival, it has been shown to mediate, togetherwith MAPK, the survival of CNS neurons promoted bythe activation of the FGFR by a synthetic peptide(Neiiendam et al., 2004). Moreover, other neurotrophicfactors can promote neuronal survival through this path-way (embryonic rat sympathetic neurons, Crowder andFreeman, 1998; Kuruvilla et al., 2000; for reviews seeBrunet et al., 2001; Rodgers and Theibert, 2002).

In this paper, we have investigated the specificityof the contributions of these pathways by analyzing theeffects of specific pharmacological inhibitors on bothwhole ciliary ganglia and dissociated neurons in culture.We provide evidence that FGFR1 is expressed in ourexperimental model. Moreover all three pathways areactivated upon bFGF stimulation and contribute to thebFGF-induced enhancement of neurite growth. On thecontrary, only the PI3-K transduction pathway mediatesthe survival-promoting effect of bFGF, by protectingneurons from apoptotic death.

MATERIALS AND METHODS

Cell Culture

Chick ciliary ganglia were dissected from E7/E8embryos and maintained in a chemically defined N2 medium(Bottenstein, 1983) as previously described (Distasi et al.,1998). Briefly, ganglia were both enzymatically [0.06% tryp-sin, in cation-free phosphate-buffered solution (PBS), for 5

min at 378C] and mechanically dissociated and resuspended inN2 medium. Nearly 15,000 cells were then plated in the mid-dle area of plastic dishes or glass coverslips coated with poly-D-lysine (PL; 100 lg/ml) and laminin (LN; 2 lg/cm2).

According to experimental protocols, human recombi-nant bFGF (20 ng/ml; Alomone Labs, Jerusalem, Israel) andthe inhibitors U73122 (0.5 lM; Calbiochem, Darmstadt, Ger-many), PD98059 (25 lM; Calbiochem), and wortmannin (10nM) were added to the medium; to maintain efficacy ofinhibitors, media containing drugs were changed every 24 hr.

CG Explant Cultures

Chick ciliary ganglia were obtained from E7/E8embryos and collected in PBS. Explants were cultured in rattail collagen gel as previously reported (Zamburlin et al.,2006) and plated in 12-multiwell plates previously treatedwith PL and LN. Briefly ganglia were taken one by one in asmall drop of PBS with a Pasteur pipette and placed in themiddle of an empty well with as little PBS as possible. A 15-ll drop of collagen gel mix was added on the top of eachganglion and kept at 378C in a CO2 incubator for 40 minuntil the gel had polymerized. Subsequently 1 ml of N2 me-dium was added to each well. When required, bFGF (20 ng/ml), U73122 (2 lM), PD98059 (25 lM), and wortmannin(10 nM) were added to the medium.

Assessment of Cell Survival

For survival assays, cells were plated on 60-mm-diame-ter/2-mm-grid culture dishes (Corning Inc., Corning, NY),and two square fields of the grid in each dish were recordedby means of a CCD video camera (PCO, Germany) con-nected to an inverted microscope (Eclipse TE 200; Nikon).Survival levels were assessed by counting neurons, in the samefield, after 24 and 48 hr of culture, and giving the values aspercentages of the counts at 2 hr. Two hours after plating,neurons were clearly distinguishable from nonneuronal cellsby their phase-bright, rounded cell bodies and short processes.We have previously shown that all birefringent, round cellsdisplay electrophysiological properties of differentiated neurons(Distasi et al., 1998) and are positive for neuronal markers(Barale et al., 1998). Each experiment was made in duplicate;three or four independent experiments were performed foreach individual treatment.

Neuronal apoptosis was assessed by labelling cells with40,6-diamidino-2-phenylindole (DAPI); after 24 and 48 hr ofculture, cells were fixed in paraformaldehyde (PAF) 4% for 20min and stained with DAPI (0.2 lg/ml) to show shrunkenand fragmented apoptotic nuclei. Double labelling with amonoclonal anti-NF68 antibody (1:300) was performed todistinguish neurons from glial cells. A minimum of five repli-cate cultures for each condition were made, and eight to tenrandom fields were analyzed at 340 magnification. Here andin neurite growth assays, the inhibitors were dissolved inDMSO (maximal dilution 1:500) which, when added alone tothe culture, had no effects.

2952 Gilardino et al.

Journal of Neuroscience Research

Immunocytochemistry

For immunocytochemical experiments, dissociated cellswere fixed in PAF 4% for 20 min at room temperature,whereas CG explants were fixed in –208C methanol for 10min. FGFR immunostaining was performed by incubatingcells for 1 hr at room temperature with a rabbit anti-FGF re-ceptor-1 antibody, raised against the extracellular region ofthe receptor (FGFR-1, 1:100). For phospho-PLCg, -ERK,and -AKT staining, cells were permeabilized with 0.1% Tri-ton-PBS and incubated at room temperature for 1 hr with aphospho-PLCg antibody (antiphospho-phospholipase Cg-1pTyr783, produced in rabbit; 1:100) or a phospho-ERK anti-body (antiphospho-ERK1 pThr202/pTyr204 and ERK2pThr185/pTyr187, developed in rabbit; 1:100) and overnightwith a phospho-AKT antibody (antiphospho-AKT Ser473 pro-duced in rabbit, Cell Signaling, Danvers, MA; 1:100).

After washing with PBS, both cells labelled with anti-FGFR and phospho-specific antibodies were incubated for 1hr at room temperature with donkey anti-rabbit IgG antibodyconjugated with the Alexa 488 fluorophore (1:100; Invitro-gen, Carlsbad, CA), washed with PBS, and mounted inDABCO (1,4-diazabicyclo[2.2.2]octane). Controls for reactiv-ity of the secondary antibody, without primary antibody incu-bation, were also performed.

Images (1,0243 1,024 pixels, 16-bit gray scale) were ana-lyzed by means of a Fluoview 200 laser scanning confocalmicroscope (Olympus America Inc., Melville, NY) with a 350oil immersion objective and acquired at an excitation wave-length of 488 nm. For each field, both the fluorescent and thedifferential interference contrast (DIC) images were captured.

For neurite outgrowth assays, both dissociated CG neu-rons and CG explants were stained with a monoclonal anti-NF68 antibody to take into account only the contribution ofneurites. Chick CG neurons and CG explants fixed, respec-tively, after 24 and 48 hr of culture were both incubatedovernight with the anti-NF68 antibody (1:300) at room tem-perature. On the following day, dissociated neurons wereincubated with an anti-mouse IgG conjugated with the Cy3fluorophore (1:1,000). In the case of organotypical cultures,the reaction was developed with a horse biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA; 1:200)detected with the peroxidase-conjugated biotin-avidin com-plex (Vector Laboratories), followed by a brief incubationwith 3,30-diaminobenzidine tetrahydrochloride (DAB; 15 mg/ml) and H2O2 (8 lg/ml).

Assessment of Neurite Outgrowth

After immunocytochemical staining, neurite outgrowthwas assessed by measuring the total area occupied by neurofi-lament-positive elements using the image processing and anal-ysis program ImageJ (Rasband, 2007) as previously described(Zamburlin et al., 2006). Briefly, for dissociated CG cultures,eight to ten fields (1,024 3 1,024 pixels, 16-bit gray scale)per culture dish were acquired by means of a Fluoview 200laser scanning confocal microscope (Olympus) with a 350 oilimmersion objective, at an excitation wavelength of 568 nm.Fields were selected by observing the DIC images and ran-domly choosing nonoverlapping fields, moving the micro-

scope stage. Contribution of neuronal somata was manuallysubtracted, and the analysis was performed by applying toeight-bit images a threshold (1–255) in order to obtain binaryimages (thresholding pixels, corresponding to neurites, arerepresented as black and background pixels are represented aswhite). Total number of neurons was evaluated from DICimages of the same fields analyzed, and these values were usedto normalize the total area occupied by black pixels (lm2).Experiments were repeated three or four times in duplicate.

In the case of CG explants, on the other hand, imageswere acquired in brightfield mode with an inverted microscopeequipped with a CCD camera (CoolSnap-Pro color RS Photo-metrics; Media Cybernetics) and an image analysis software(Image Pro Plus 4.1 for Windows; Media Cybernetics). Aftersubtraction of the area corresponding to the body of the wholeexplant, previously reconstructed from a collage of partialimages (1,392 3 1,040 pixels, eight-bit gray scale), binaryimages were obtained again by applying the 1–255 threshold.The percentage of black square pixels over the total area (2,0003 2,000 pixels) was used as an index of global neurite growth.Data are the average of three to seven independent experiments,and five to twelve explants per condition were analyzed.

Evaluation of Target PhosphorylationDownstream of FGFR

Dissociated CG neurons were plated for 2 hr on PL- andLN-coated glass coverslips, incubated for 1 hr with N2 me-dium, and then treated with or without bFGF 20 ng/ml for 15,30, and 60 min. Cells were then fixed and stained with the anti-phospho-antibodies (antiphospho-PLCg, -ERK, and -AKT)specific for the three pathways analyzed. For each of them, thehighest level of activation was chosen as the time correspondingto maximal staining intensity, and at this time point the inhibi-tory activities of PD98059 and wortmannin, preincubated for15 min, were evaluated. Confocal images (1,0243 1,024 pixels,16-bit gray scale), taken at the plane of neuronal cell bodieswere then analyzed in ImageJ software. Average fluorescenceintensity within regions of interest (ROIs), covering single cellbodies, was calculated as the sum of the gray values of all thepixels in each ROI divided by the number of pixels (ROI area).

For each condition, all values from tens of individualcells from four fields of at least three experiments in duplicate(for a total of 150–300 cells per condition) were pooled, andmean and SE were calculated from the pooled data. For everydish, 20 glial cells were selected at random, and the fluores-cence intensities were averaged; these values were comparedamong all dishes (both in the presence of bFGF alone andwith the inhibitor added), and the values were found to benot significantly different.

Statistical Analysis

Data are expressed as mean 6 SE. Statistical analyses ofcontrol and experimental groups were performed in SPSS ver-sion 14.0 for Windows (SPSS Inc., Chicago, IL). Unless oth-erwise stated, one-way ANOVA was performed to analyzeexperiments, and Bonferroni post hoc tests were used to eval-uate statistically significant differences among the groups. Stu-dent’s t-test was used to evaluate statistical differences between

Effects of bFGF in Ciliary Ganglion Neurons 2953


means of apoptotic neurons, using 0 as a test value. If not oth-erwise specified, all chemicals and drugs were purchased fromSigma Chemical Co. (St. Louis, MO).

RESULTS

FGFR1 Expression and Localization

Because no data on the specific expression and local-ization of FGFRs in chick ciliary ganglion neurons havebeen previously reported, we performed immunocytochem-ical experiments on dissociated cultures with an anti-FGFR1antibody. As assessed by comparing fluorescent images withthe corresponding DIC fields, anti-FGFR1 antibody mark-edly labelled 97% of CG neurons (white arrows in Fig.1A,C show an example of an unlabelled neuron).

As reported in Figure 1, the antibody stained bothsomata and neuritic processes of CG neurons (see insetin Fig. 1B); somatic staining is localized in the peripheryand cytosol, not in nuclei. In addition, some neuronsdisplay a slightly enhanced staining at the membranelevel (see inset in Fig. 1A).

FGFR1 immunopositivity was also observed onglial cells (95%), even if the staining was less intense(white stars in Fig. 1B,C indicate representative FGFR1-immunopositive glial cells). Figure 1D,E shows absenceof immunoreactivity when cells were incubated with thesecondary antibody alone.

Involvement of the PLCc, MAPK, and PI3-KPathways in Neuronal Survival Induced by bFGF

Neuronal survival in dissociated cultures was eval-uated at 24 and 48 hr, and the involvement of the threepathways in the bFGF-promoted survival was assessed bycomparing cultures with and without the specific inhibi-tors. Cultures were maintained in the presence of PLand LN, either with or without added bFGF. The useof LN as a control, a widely used protocol (see, e.g.,Williams et al., 1994; Lom et al., 1998; Webber et al.,

2005), was required by the fact that in its absence, aspreviously reported (Distasi et al., 1998), neuronal cellstended to aggregate and to form clusters, preventing aquantitative assessment of cell number. Even in the pres-ence of PL and LN, after 48 hr significant clustering

Fig. 1. Expression and localization of FGFR1 in dissociated CG cul-tures. A: Confocal image taken at the plane of neuronal cell bodiesof dissociated CG cultures stained with the anti-FGFR1 antibody 24hr after plating. An example of an unstained neuron is marked withan arrow. Inset shows a typical labelled neuron. B: Image of thesame field as in A but taken at the plane of neurites and glial cells;examples of labelled glial cells are marked with stars. Inset illustratesthe staining along neurites. C: The corresponding DIC image of Aand B, with the same symbols as previously used. D,E: Fluorescenceand DIC images of the same field showing control for aspecific stain-ing of the secondary anti-rabbit antibody. Scale bar 5 20 lm. [Colorfigure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

Fig. 2. Only the PI3-K pathway is involved in the survival-promot-ing effect of bFGF on CG neurons. A: The PLCg inhibitor U73122(U; 0.5 lM) had no effect on neuronal survival up to 48 hr eitherunder control conditions (U0.5 after 24 hr: 77.5% 6 3.7%, after 48hr: 61.9% 6 3.7%; CTRL after 24 hr: 70.4% 6 2.2%, after 48 hr:53.2% 6 3.2%) or when bFGF (20 ng/ml) was added to the extrac-ellular medium (bFGF 1 U0.5 after 24 hr: 91.7% 6 3.2%, after 48hr: 95.1% 6 2.9%; bFGF after 24 hr: 90.6% 6 1.6%, after 48 hr:89.9% 6 1.7%). **P < 0.01 vs. CTRL (24 hr); 11P < 0.01 vs.CTRL (48 hr). B: PD98059 (PD; 25 lM), an inhibitor of MEK,had no effect on neuronal survival promoted by bFGF either after 24hr (bFGF 1 PD25: 91.6% 6 1.2%; bFGF: 95.8% 6 1.2%) or after48 hr (bFGF 1 PD25: 91.4% 6 1.0%; bFGF: 96.9% 6 0.6%), butsignificantly reduced survival under control conditions both at 24 hr(PD25: 52.4% 6 1.5%; CTRL: 64.9% 6 2.5%) and at 48 hr (PD25:28.7% 6 1.6%; CTRL: 49.4% 6 1.9%). **P < 0.01 vs. CTRL (24hr); 11P < 0.01 vs. CTRL (48 hr). C: Wortmannin (WM; 10 nM),which blocks the PI3-K pathway, significantly reduced bFGF-induced neuronal survival at 48 hr (bFGF 1 WM10 after 24 hr:94.9% 6 0.5%, after 48 hr: 64.7% 6 4.9%; bFGF after 24 hr: 95.4%

6 1.6%, after 48 hr: 98.9% 6 0.4%), without affecting survival undercontrol conditions (WM10 after 24 hr: 64.1% 6 1.8%, after 48 hr:42.9% 6 3.8%; CTRL after 24 hr: 58.7% 6 4.6%, after 48 hr:41.9% 6 3.4%). **P < 0.01 vs. CTRL (24 hr); 11P < 0.01 vs.CTRL (48 hr); ##P < 0.01 vs. bFGF (48 hr). Values reported in A–C represent mean 6 SE of three or four independent experimentsperformed in duplicate. D: Block of the PI3-K pathway by wort-mannin (WM; 10 nM) induced apoptosis in a fraction of CG neu-rons cultured for 48 hr in the presence of bFGF. Left, phase-contrastimages; right, corresponding images of cultures stained with the nu-clear marker DAPI (blue) and with the anti-NF68 antibody (red). Inthe presence of wortmannin for 48 hr (lower panel), fragmented neu-ronal nuclei can be detected (arrow). No fragmentation can benoticed either under control conditions (upper panel) or after 24 hrwith WM (middle panel). E: Percentages of apoptotic cells after 48hr in culture under control conditions and with bFGF, with or with-out 10 nM wortmannin. Values are reported as mean 6 SE of atleast five replicate cultures for each condition. t-test: **P < 0.01 vs.bFGF. Scale bar 5 10 lm.

"



occurred; for this reason, survival ratios were not eval-uated at longer times.

In agreement with previous data (Distasi et al.,1998), in the presence of PL and LN (CTRL), the per-centage of surviving neurons was about 65% at 24 hr,with a further reduction to 48% at 48 hr; when 20 ng/

ml bFGF was added to the culture medium, neuronalsurvival was markedly increased to 93% at 24 hr and95% at 48 hr (percentages obtained by pooling the dataon CTRL and bFGF from the three sets of experimentsperformed with the inhibitors; see Fig. 2A–C). We sub-sequently tested the three inhibitors at different concen-

Figure 2.



trations (data not shown) and selected those at which noeffect could be observed under control conditions (butsee below for the MEK/MAPKK inhibitor).

The PLCg inhibitor U73122 at the concentrationof 0.5 lM had no effect on neuronal survival ratios (Fig.2A) either in the presence of LN alone (CTRL) orwhen 20 ng/ml bFGF was added. PD98059, a MEK in-hibitor, was used at the concentration of 25 lM; at thisconcentration, no effect could be detected on bFGF-induced neuronal survival, at either 24 hr or 48 hr;interestingly, a small but significant reduction of survivalin the presence of only LN was observed at both 24 hrand 48 hr, pointing to a role of this pathway in the sur-vival promoted by LN (Fig. 2B).

At a concentration of 10 nM, the PI3-K inhibitorwortmannin had no effect on LN-induced neuronal sur-vival; in this case, however, in the presence of 20 ng/mlbFGF, the inhibitor, though not effective at 24 hr, sig-nificantly reduced bFGF-induced survival at 48 hr (Fig.2C). This pathway, therefore, appears to be the onlyone, among the three tested, involved in the bFGF-induced promotion of neuronal survival in our experi-mental model; to clarify the mechanism involved in thistrophic effect, we assessed apoptotic death ratios in neu-rons cultured under the four conditions (CTRL andbFGF, with and without wortmannin). Apoptotic eventsin the neuronal population were detected by doublestaining with the nuclear marker DAPI and an anti-NF68 antibody (Fig. 2D,E). Figure 2E shows that, undercontrol conditions, some neurons undergo nuclear frag-mentation after 48 hr in culture (9.4% 6 1.2%); the per-centage is not significantly increased by the presence of10 nM wortmannin (14.1% 6 1.5%); in the presence ofbFGF, apoptosis is virtually absent but increases to 8.2%6 1.7% in the presence of the inhibitor, pointing to anantiapoptotic effect of the bFGF-activated PI3-K path-way on these neurons. At 24 hr in the presence of theinhibitor, no nuclear fragmentation was observed in neu-rons (Fig. 2D, middle); this finding is relevant, rulingout an involvement of cell death in the effect of wort-mannin on bFGF-induced neurite growth at 24 hr,described in the following section.

Involvement of the PLCc, MAPK, and PI3-KPathways in the Promotion of Neurite Growth

Following the protocols developed in a previouspaper (Zamburlin et al., 2006), we performed a quantita-tive evaluation of global neurite growth on both dissoci-ated neurons in culture and whole ganglia explants. Aspreviously described, on whole ganglia, neurite growthwas assessed at 48 hr of culture; on dissociated neurons,measurements were taken after 24 hr, because at longertimes neuronal aggregation and neurite fasciculationmade it very difficult to obtain reliable data.

U73122, at the concentration of 0.5 lM, signifi-cantly reduced bFGF-induced neurite growth in dissoci-ated cultures, without affecting growth under controlconditions, in the sole presence of the adhesion mole-

cules (Fig. 3A). Global occupation values are representedin Figure 3B.

On whole ganglia, 0.5 lM U73122 had no effectin all conditions; however, a slightly higher concentra-tion, 2 lM, induced a significant reduction in neuritegrowth promoted by bFGF without affecting basalgrowth in LN (CTRL; Fig. 3C,D) and without affectingeither the morphology or dimensions of the ganglia. Asshown in Figure 4, PD98059 (25 lM) induced similarand significant reductions of bFGF-induced neuritegrowth on both dissociated neurons and whole ganglia;no significant effects were observed either in dissociatedcells or in whole ganglia under control conditions (Fig.4B,D).

Similar results were obtained with wortmannin; atthe concentration of 10 nM, the inhibitor did not haveany effect on global neurite growth in dissociated cul-tures in the presence of LN only, while significantlyreducing bFGF-induced growth (see Fig. 5A,B). Similarresults were obtained on whole ganglia (Fig. 5C,D).

bFGF-Induced Phosphorylation of the PLCc,MAPK, and PI3-K Pathways

To confirm that bFGF specifically activates thethree signalling pathways previously shown to beinvolved in the neurotrophic effects promoted by thisfactor on CG neurons, we analyzed the phosphorylationof PLCg, ERK, and AKT by using the correspondingantiphospho-antibodies. All three enzymes showed a ba-sal level of activation under control conditions, whichsignificantly increased following bFGF treatment (Fig.6A,C,E). Moreover, in all cases, glial cells were weaklystained by the antibodies used, either with or withoutbFGF (see Fig. 6F). Time course analysis of immunola-belled bFGF-treated neurons (data not shown) revealeddifferent peaks of activation for the three pathways: max-imal PLCg activation occurred after 60 min of bFGFexposure, whereas pERK and pAKT both reached theirfluorescence peak within 30 min (Fig. 6B,D,F). In thecase of ERK and AKT, application of the kinase inhibi-tors PD98059 and wortmannin, respectively, effectivelyprevented bFGF-induced phosphorylation of the down-stream targets (Fig. 6C–F).

DISCUSSION

In this study, by means of a pharmacologicalapproach, we have dissected the specific contributions ofthe three main signal transduction pathways activated bybFGF in a classical model of developing parasympatheticnerve cells, ciliary ganglion neurons. We show that themost widely expressed FGF receptor, FGFR1, is presentin nearly all neuronal cells, both in the somata and alongneurites and to a lesser extent in glial cells. The latterobservation raises the issue of a possible dependence ofthe observed neuronal responses from glial cell activationon bFGF. We have previously shown (Zamburlin et al.,2006) that this potent pleiotropic factor promotes neuritegrowth through a mechanism that acts, at least in part,



directly on neurons; similar findings have been reportedfor neuronal survival (Unsicker et al., 1992) and havebeen confirmed in our culture conditions (unpublishedresults). Therefore, even if we cannot exclude that theblockers we have employed may interfere with glial cell-derived signals, the data presented here point to a role ofthe neuronal PLCg, MAPK, and PI3-K cascades in con-trolling two key parameters in the development of para-sympathetic nerves.

Activation of FGFR1 actually increased the phos-phorylation of all three enzymes above the levelsobserved in the presence of LN alone, as evidenced by

immunocytochemical observations. It must be notedthat, in the case of ERK, the enzyme was alreadystrongly phosphorylated under this control condition;this may be related to the finding that inhibition of theMEK-ERK pathway reduces LN-promoted neuronalsurvival. Moreover, the lower levels of phosphorylationobserved for all three enzymes in glial cells give furthersupport to the hypothesis that glial cell-mediated effectsof bFGF are not the main determinant of neuronalresponses in this model.

For two of the targets investigated, ERK/MAPKand AKT, it was possible to show that the pharmacological

Fig. 3. The PLCg pathway is involved in the growth of neurites ofCG neurons promoted by bFGF. A: Binary images of neuritesstained with the anti-NF68 antibody at 24 hr of culture, after manualremoval of the areas occupied by cell bodies. Dissociated culturedneurons under control conditions and with 20 ng/ml bFGF added,with or without the PLC blocker U73122 (U; 0.5 lM), are com-pared. B: Quantification of neurite outgrowth as the area occupiedby black pixels, in the four experimental conditions of A. Each valuerepresents mean 6 SE of three or four replicates performed in dupli-cate (CTRL: 417 6 24 lm2; bFGF: 642 6 36 lm2; U0.5: 358 615 lm2; bFGF 1 U0.5: 296 6 28 lm2). **P < 0.01 vs. CTRL;

11P < 0.01 vs. bFGF. C: CG explants embedded in a collagen geland stained with the anti-NF68 antibody after 48 hr in culture, inthe four experimental conditions as in A. D: Quantitative assessmentof the area occupied by neurites in the explants (percentage of blacksquare pixels over the entire area, after subtraction of the area corre-sponding to the body of the whole explant) under control conditionsand with bFGF added, with or without 2 lM U73122 (U; 2 lM).Data are reported as mean 6 SE of four to twelve explants per condi-tion analyzed (CTRL: 20.8% 6 2.8%; bFGF: 40.8% 6 1.2%; U2:16.4% 6 1.5%; bFGF 1 U2: 26.3% 6 4.3%). **P < 0.01 vs. CTRL;11P < 0.01 vs. bFGF. Scale bars 5 50 lm in A; 400 lm in C.



inhibitors effectively reduced phosphorylation levels. Forthe third one, PLCg, more complex protocols wouldhave been required, such as assays of phosphoinositidemetabolism, that were outside the scope of this paper.Interestingly, our data point to a specific role of thePI3-K enzyme in bFGF-induced neuronal survival, inaccordance with the findings of other authors(Neiiendam et al., 2004); on the other hand, all threepathways seem to be involved in the promotion of neu-rite growth.

The strength of these findings is supported by theparallel use of dissociated cultures and whole-gangliaexplants and can be evidenced by using inhibitor con-

centrations that are in the range commonly reported inthe literature but low enough to avoid possible aspecificeffects. For the PLCg inhibitor U73122, a slightly higherdose was necessary to observe a reduction in neuritegrowth in whole ganglia compared with dissociated cul-tures; the fact that in an organotypic preparation theeffective dose of pharmacological agents may be higherthan that on isolated neurons has been reported in otherpapers (see, e.g., Webber et al., 2005). On the otherhand, we have already provided evidence (Zamburlinet al., 2006) that, with our experimental protocols, theembedding of whole ganglia in collagen does not pre-vent either the sensing of extracellular matrix molecules

Fig. 4. Involvement of the MAPK pathway in bFGF-induced neuritegrowth. A: Binary images of neurites stained with the anti-NF68antibody at 24 hr of culture. Dissociated cultured neurons under con-trol conditions and with 20 ng/ml bFGF added, with or without theMAPKK (MEK) blocker PD98059 (PD; 25 lM) are compared. B:Quantification of neurite outgrowth in the four experimental condi-tions of A. Each value represents mean 6 SE of three or four repli-cates performed in duplicate (CTRL: 463 6 30 lm2; PD25: 426 626 lm2; bFGF: 645 6 32 lm2; bFGF 1 PD25: 493 6 25 lm2).**P < 0.01 vs. CTRL; 11P < 0.01 vs. bFGF. C: CG explants em-

bedded in a collagen gel and stained with the anti-NF68 antibody af-ter 48 hr in culture, in the four experimental conditions as in A. D:Quantitative assessment of the area occupied by neurites in theexplants under the four experimental conditions. Data are reported asmean 6 SE of six to twelve explants analyzed for each condition(CTRL: 12.4% 6 2.1%; PD25: 6.1% 6 0.9%; bFGF: 24.5% 63.5%; bFGF 1 PD25: 10.3% 6 1.9%). **P < 0.01 vs. CTRL; 11P< 0.01 vs. bFGF. Technical details are as for Figure 3. Scale bars 550 lm in A; 400 lm in C.



or the diffusion of the neurotrophic factor. The resultsobtained with the two models are indeed highly coher-ent and reinforce the conclusions that can be drawnfrom our data.

In accordance with the protocols used by otherauthors (Lom et al., 1998; Webber et al., 2005), wechose to analyze the effects of the three inhibitors onbFGF-induced neuronal survival and neurite growth bytaking as controls cultures maintained in the presence ofPL and LN. In our experimental model, this protocol isparticularly mandatory, because, without a strong sub-strate of adhesion molecules, ciliary ganglion neurons inthe presence of bFGF can survive for many days but

tend to aggregate in big clusters connected by highly fas-ciculated neurite bundles (Distasi et al., 1998), thus pre-venting any quantitative analysis of specific parameters.This choice, on the other hand, poses significant chal-lenges, insofar as it is known that adhesion moleculesand their integrin receptors have a role on their own inpromoting survival and neurite growth and do it by acti-vating signal transduction pathways that are at least inpart shared with neurotrophic factors (Perron and Bixby,1999; Tucker et al., 2005). For these reasons, whenassessing the involvement of specific pathways by meansof pharmacological tools, careful attention must be paidto select concentrations at which the effects of the

Fig. 5. Involvement of the PI3-K pathway in neurite growth pro-moted by bFGF. A: Binary images of neurites stained with the anti-NF68 antibody at 24 hr of culture. Dissociated cultured neuronsunder control conditions and with 20 ng/ml bFGF added, with orwithout the PI3-K blocker wortmannin (WM; 10 nM), are com-pared. B: Quantification of neurite outgrowth in the four experi-mental conditions of A. Each value represents mean 6 SE of threeor four replicates performed in duplicate (CTRL: 511 6 14 lm2;WM10: 484 6 21 lm2; bFGF: 662 6 37 lm2; bFGF 1 WM10:473 6 17 lm2). **P < 0.01 vs. CTRL; 11P < 0.01 vs. bFGF. C:

CG explants embedded in a collagen gel and stained with the anti-NF68 antibody after 48 hr in culture, under the four experimentalconditions as in A. D: Quantitative assessment of the area occupiedby neurites in the explants under the four experimental conditions.Data are reported as mean 6 SE of 12 ganglia analyzed for each con-dition (CTRL: 17.3% 6 1.9%; bFGF: 37.7% 6 2.2%; WM10:13.5% 6 1.5%; bFGF 1 WM10: 28.1% 6 2.6%). **P < 0.01 vs.CTRL; 1P < 0.05 vs. bFGF. Technical details are as for Figure 3.Scale bars 5 50 lm in A; 400 lm in C.



Fig. 6. Quantitative immunocytochemical analysis of phopsho-PLCg, -ERK, and -AKT. All three pathways analyzed are enhancedin CG neurons upon treatment with 20 ng/ml bFGF. A: Applicationof bFGF for 60 min significantly increased the level of phospho-PLCg. **P < 0.01 vs. CTRL. B: Examples of confocal images ofphospho-PLCg-labelled neurons, with the corresponding DIC fields;after 60 min of bFGF treatment, an enhancement in labelling was de-tectable. C: In the presence of bFGF for 30 min, phospho-ERK av-erage fluorescence intensity was significantly higher than under con-trol conditions, and the increase in fluorescence was completely abol-ished by treatment with PD98059 25 lM. **P < 0.01 vs. CTRL;11P < 0.01 vs. bFGF. D: Sample fields showing a weak stainingunder control conditions, an increased labelling after bFGF treatment,

and a reduced staining in presence of bFGF with MEK inhibitor. E:bFGF significantly increased the level of phospho-AKT fluorescenceintensity after 30 min of treatment, whereas incubation with wort-mannin 10 nM prevented the bFGF-induced increase in phosphoryl-ation. **P < 0.01 vs. CTRL; 11P < 0.01 vs. bFGF. F: Examples ofphospho-AKT stained CG neurons under control conditions showingweak staining; bFGF promoted an increment in fluorescence. In thepresence of wortmannin, a clear reduction was evident. Values inA,C,E represent mean 6 SE obtained by pooling tens of individualcells from four fields of at least three experiments in duplicate (for atotal of 150–300 cells per condition). Scale bar 5 20 lm. [Color fig-ure can be viewed in the online issue, which is available at www.interscience.wiley.com.]


neurotrophic factor can be clearly separated by those de-pendent on integrin signalling.

In this context, it must be mentioned that a paperby Peterziel et al. (2002) reported that interfering witheither the MAPK or the PI3-K pathway resulted in asignificant reduction of the bFGF-induced neuronal sur-vival in the same experimental model as used in this pa-per, E8 chick ciliary ganglion neurons, at 24 hr of cul-ture. This is in apparent conflict with our findings,which point to an involvement of PI3-K only, and at alater time (48 hr), with no contribution of the MAPKpathway. However, two significant differences existbetween the protocols we used to obtain the datareported above and those employed by Peterziel andcolleagues: 1) the survival ratios at 24 hr with 10 ng/mlbFGF (apparently about 30%, taking into account thecell number at seeding and the area considered for thecell counts) were much lower than those (>90%) wehave reported in this and in previous papers (Distasiet al., 1998; Zamburlin et al., 2006), pointing to somerelevant differences in the culture conditions that mighthave biased the survival ratios; 2) even if the neuronswere plated on LN-covered dishes, no evaluation of theeffect of the inhibitors on LN-dependent cell survivalwas performed, preventing a precise evaluation of thespecific effect of the inhibitors on the pathways activatedby bFGF. The very high survival ratios we observed inthe presence of bFGF and the finding that the MEK in-hibitor reduces neuronal survival already at 24 hr in thepresence of LN only, while it has no effect on bFGF-induced survival, may be the key to understanding thediscrepancies between the data presented here and thoseby Peterziel et al. (2002). It has been shown that theMAPK pathway is crucial for LN-mediated neuronalsurvival, and its block has relevant effects, even on atime scale of days (Perron and Bixby, 1999); on the con-trary, the robust bFGF-induced survival is dependentpredominantly on other signalling cascades (such as thePI3-K one) and, consequently, not significantly affectedby the blockade of ERK-MAPK, at least for the timestaken into account in this work.

The selective role of the PI3-K enzyme in control-ling bFGF-induced neuronal survival is in agreementwith data obtained with other neurotrophic factors (see,e.g., Rodgers and Theibert, 2002); the fact that its actionapparently depends on a protective effect on apoptoticdeath observed at 48 hr of culture is of relevance, in thelight of the huge programmed death of ciliary ganglionneurons that occurs in vivo between E8 and E14, bring-ing the original neuronal population of postmitotic neu-rons to nearly one-half (Dryer, 1994). It must be noted,however, that, after 24 hr in the presence of bFGF andthe PI3-K inhibitor, no apoptotic neurons could bedetected, thus ruling out any contribution of cell deathto the observed reduction of neurite growth by thispharmacological agent.

On the other hand, the finding that all three sig-nalling cascades are involved in the enhancement of neu-rite growth is of particular interest, because, in other ex-

perimental models, not all of them seem to play a rolein this context (see, e.g., Webber et al., 2005). It couldbe of interest to know whether all of them converge onthe same targets and control the same parameters ofgrowth, such as rate of elongation, number of neurites,and orientation. More detailed experiments are needed,and they will be undertaken in future work.

ACKNOWLEDGMENTS

We thank Susanna Antoniotti and Simona Dal-mazzo for helpful comments and advice.

REFERENCES

Abe K, Saito H. 2000. Neurotrophic effect of basic fibroblast growth fac-

tor is mediated by the p42/p44 mitogen-activated protein kinase cas-

cade in cultured rat cortical neurons. Brain Res Dev Brain Res 122:

81–85.

Abe K, Saito H. 2001. Effects of basic fibroblast growth factor on central

nervous system functions. Pharmacol Res 43:307–312.

Abe K, Aoyagi A, Saito H. 2001. Sustained phosphorylation of mitogen-

activated protein kinase is required for basic fibroblast growth factor-

mediated axonal branch formation in cultured rat hippocampal neurons.

Neurochem Int 38:309–315.

Barale E, Torre M, Haimann C, Lovisolo D. 1998. IGF-I enhances sur-

vival of embryonic chick ciliary ganglion neurons in a calcium-depend-

ent way. Neuroreport 9:2513–2517.

Bottenstein JE. 1983. Defined media for dissociated neural cultures. Curr

Methods Cell Neurobiol 4:107–130.

Brunet A, Datta SR, Greenberg ME. 2001. Transcription-dependent and

-independent control of neuronal survival by the PI3K-Akt signaling

pathway. Curr Opin Neurobiol 11:297–305.

Crowder RJ, Freeman RS. 1998. Phosphatidylinositol 3-kinase and Akt

protein kinase are necessary and sufficient for the survival of nerve

growth factor-dependent sympathetic neurons. J Neurosci 18:2933–

2943.

Dailey L, Ambrosetti D, Mansukhani A, Basilico C. 2005. Mechanisms

underlying differential responses to FGF signaling. Cytokine Growth

Factor Rev 16:233–247.

Desire L, Courtois Y, Jeanny JC. 2000. Endogenous and exogenous

fibroblast growth factor 2 support survival of chick retinal neurons by

control of neuronal bcl-xL and bcl-2 expression through a fibroblast

growth factor receptor 1- and ERK-dependent pathway. J Neurochem

75:151–163.

Distasi C, Torre M, Antoniotti S, Munaron L, Lovisolo D. 1998. Neuro-

nal survival and calcium influx induced by basic fibroblast growth factor

in chick ciliary ganglion neurons. Eur J Neurosci 10:2276–2286.

Doherty P, Walsh FS. 1996. CAM-FGF receptor interactions: a model

for axonal growth. Mol Cell Neurosci 8:99–111.

Dreyer D, Lagrange A, Grothe C, Unsicker K. 1989. Basic fibroblast

growth factor prevents ontogenetic neuron death in vivo. Neurosci

Lett 99:35–38.

Dryer SE. 1994. Functional development of the parasympathetic neurons

of the avian ciliary ganglion: a classic model system for the study of

neuronal differentiation and development. Prog Neurobiol 43:281–322.

Eswarakumar VP, Lax I, Schlessinger J. 2005. Cellular signaling by fibro-

blast growth factor receptors. Cytokine Growth Factor Rev 16:139–

149.

Ferrari G, Minozzi MC, Toffano G, Leon A, Skaper SD. 1989. Basic

fibroblast growth factor promotes the survival and development of mes-

encephalic neurons in culture. Dev Biol 133:140–147.

Ford-Perriss M, Abud H, Murphy M. 2001. Fibroblast growth factors in

the developing central nervous system. Clin Exp Pharmacol Physiol 28:

493–503.



Gilardino A, Perroteau I, Lovisolo D, Distasi C. 2000. In vitro identifica-

tion of dividing neuronal precursors from chick embryonic ciliary gan-

glion. Neuroreport 11:1209–1212.

Grothe C, Timmer M. 2007. The physiological and pharmacological role

of basic fibroblast growth factor in the dopaminergic nigrostriatal sys-

tem. Brain Res Rev 54:80–91.

Grothe C, Wewetzer K, Lagrange A, Unsicker K. 1991. Effects of basic

fibroblast growth factor on survival and choline acetyltransferase devel-

opment of spinal cord neurons. Brain Res Dev Brain Res 62:257–261.

Grothe C, Haastert K, Jungnickel J. 2006. Physiological function and pu-

tative therapeutic impact of the FGF-2 system in peripheral nerve

regeneration—lessons from in vivo studies in mice and rats. Brain Res

Rev 51:293–299.

Hall H, Williams EJ, Moore SE, Walsh FS, Prochiantz A, Doherty P.

1996. Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and

neurite outgrowth by a cell-membrane permeable phosphopeptide.

Curr Biol 6:580–587.

Haynes LW. 1988. Fibroblast (heparin-binding) growing factors in neu-

ronal development and repair. Mol Neurobiol 2:263–289.

Hossain WA, Morest DK. 2000. Fibroblast growth factors (FGF-1, FGF-

2) promote migration and neurite growth of mouse cochlear ganglion

cells in vitro: immunohistochemistry and antibody perturbation. J Neu-

rosci Res 62:40–55.

Katsuki H, Itsukaichi Y, Matsuki N. 2000. Distinct signaling pathways

involved in multiple effects of basic fibroblast growth factor on cultured

rat hippocampal neurons. Brain Res 885:240–250.

Klint P, Claesson-Welsh L. 1999. Signal transduction by fibroblast

growth factor receptors. Front Biosci 4:D165–D177.

Kuruvilla R, Ye H, Ginty DD. 2000. Spatially and functionally distinct

roles of the PI3-K effector pathway during NGF signaling in sympa-

thetic neurons. Neuron 27:499–512.

Lom B, Hopker V, McFarlane S, Bixby JL, Holt CE. 1998. Fibroblast

growth factor receptor signaling in Xenopus retinal axon extension. J

Neurobiol 37:633–641.

Neiiendam JL, Køhler LB, Christensen C, Li S, Pedersen MV, Ditlevsen

DK, Kornum MK, Kiselyov VV, Berezin V, Bock E. 2004. An

NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neu-

rite outgrowth and neuronal survival in primary rat neurons. J Neuro-

chem 91:920–935.

Oh DY, Park SY, Cho JH, Lee KS, Min Do S, Han JS. 2007. Phospho-

lipase D1 activation through Src and Ras is involved in basic fibroblast

growth factor-induced neurite outgrowth of H19-7 cells. J Cell Bio-

chem 101:221–234.

Perron JC, Bixby JL. 1999. Distinct neurite outgrowth signaling path-

ways converge on ERK activation. Mol Cell Neurosci 13:362–378.

Peterziel H, Unsicker K, Krieglstein K. 2002. TGFbeta induces GDNF

responsiveness in neurons by recruitment of GFRalpha1 to the plasma

membrane. J Cell Biol 159:157–167.

Rasband WS. 2008. ImageJ. http://rsb.info.nih.gov/ij/.

Rodgers EE, Theibert AB. 2002. Functions of PI 3-kinase in develop-

ment of the nervous system. Int J Dev Neurosci 20:187–197.

Temple S, Qian X. 1995. bFGF, neurotrophins, and the control or corti-

cal neurogenesis. Neuron 15:249–252.

Tsai PS, Werner S, Weiner RI. 1995. Basic fibroblast growth factor is a

neurotropic factor in GT1 gonadotropin-releasing hormone neuronal

cell lines. Endocrinology 136:3831–3838.

Tucker BA, Rahimtula M, Mearow KM. 2005. Integrin activation and

neurotrophin signaling cooperate to enhance neurite outgrowth in sen-

sory neurons. J Comp Neurol 486:267–280.

Unsicker K, Reichert-Preibsch H, Schmidt R, Pettmann B, Labourdette

G, Sensenbrenner M. 1987. Astroglial and fibroblast growth factors

have neurotrophic functions for cultured peripheral and central nervous

system neurons. Proc Natl Acad Sci USA 84:5459–5463.

Unsicker K, Reichert-Preibsch H, Wewetzer K. 1992. Stimulation of

neuron survival by basic FGF and CNTF is a direct effect and not

mediated by nonneuronal cells: evidence from single cell cultures. Brain

Res Dev Brain Res 65:285–288.

Walicke P, Cowan WM, Ueno N, Baird A, Guillemin R. 1986. Fibro-

blast growth factor promotes survival of dissociated hippocampal neu-

rons and enhances neurite extension. Proc Natl Acad Sci USA 83:

3012–3016.

Webber CA, Chen YY, Hehr CL, Johnston J, McFarlane S. 2005. Mul-

tiple signaling pathways regulate FGF-2-induced retinal ganglion cell

neurite extension and growth cone guidance. Mol Cell Neurosci 30:

37–47.

Westermann R, Grothe C, Unsicker K. 1990. Basic fibroblast growth

factor (bFGF), a multifunctional growth factor for neuroectodermal

cells. J Cell Sci Suppl 13:97–117.

Williams EJ, Doherty P. 1999. Evidence for and against a pivotal role of

PI 3-kinase in a neuronal cell survival pathway. Mol Cell Neurosci 13:

272–280.

Williams EJ, Furness J, Walsh FS, Doherty P. 1994. Characterisation of

the second messenger pathway underlying neurite outgrowth stimulated

by FGF. Development 120:1685–1693.

Zamburlin P, Gilardino A, Dalmazzo S, Ariano P, Lovisolo D. 2006.

Temporal dynamics of neurite outgrowth promoted by basic fibroblast

growth factor in chick ciliary ganglia. J Neurosci Res 84:505–514.



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